Anti-EGFRvIII antibodies and uses thereof

ABSTRACT

The present invention provides antibodies that bind to the class III variant of EGFR (EGFRvIII) and methods of using the same. According to certain embodiments, the antibodies of the invention bind human EGFRvIII with high affinity. The antibodies of the invention may be fully human antibodies. The invention includes anti-EGFRvIII antibodies conjugated to a cytotoxic agent, radionuclide, or other moiety detrimental to cell growth or proliferation. The antibodies of the invention are useful for the treatment of various cancers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/643,886, filed Mar. 10, 2015, which claims the benefit under 35U.S.C. § 119(e) of U.S. Provisional Application No. 61/950,963, filed onMar. 11, 2014, the disclosure of which is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to human antibodies and antigen-bindingfragments of human antibodies that specifically bind the deletionmutants of human epidermal growth factor receptor (EGFR), in particular,the class III deletion mutant, EGFRvIII, and therapeutic and diagnosticmethods of using those antibodies.

BACKGROUND

Overexpression and/or gene amplification of the epidermal growth factor(EGF) receptor, or EGFR, have been reported in multiple human tumors,including those in breast, ovarian, bladder, brain, and various squamouscarcinomas (Wong, A. J. et al., 1987, Proc. Natl. Acad. Sci. USA,84:6899-6903; Harris et al., 1992, Natl. Cancer Inst. Monogr.11:181-187). However, targeting the EGFR as an anti-neoplastictherapeutic method has been problematic as many normal tissues alsoexpress this receptor and may get targeted along with the neoplastictargets. Meanwhile, it has been reported that many glioblastomas havingEGFR gene amplification frequently contain gene rearrangement (Ekstrand,A. J. et al., 1992, Proc. Natl. Acad. Sci. USA, 89:4309-4313; Wong A. J.et al., 1992, Proc. Natl. Acad. Sci. USA, 89:2965-2969). In one study,17 out of 44 glioblastomas were found to have one or more alterations inthe EGFR coding sequence and all of these cases contained amplifiedEGFR, while none of the 22 cases without gene amplification showed anytumor-specific sequence abnormalities (Frederick, L. et al., 2000,Cancer Res 60:1383-1387). The same study also showed that multiple typesof EGFR mutations could be detected in individual tumors.

The class III variant of the EGFR (EGFRvIII) is the most frequentlyfound EGFR variant in glioblastoma (Bigner et al., 1990, Cancer Res50:8017-8022; Humphrey et al., 1990, Proc Natl Acad Sci USA87:4207-4211; Yamazaki et al., 1990, Jap J Cancer Res 81:773-779;Ekstrand et al., 1992, Proc Natl Acad Sci USA 89:4309-4313; Wikstrand etal., 1995, Cancer Res 55:3140-3148; and Frederick et al., 2000, CancerRes 60:1383-1387). EGFRvIII is characterized by a deletion of exons 2-7of the EGFR gene, resulting in an in-frame deletion of 801 base pairs ofthe coding region, i.e., deletion of 6-273 amino acid residues (based onthe residue numbers of mature EGFR), as well as the generation of a newglycine at the fusion junction (Humphrey et al., 1988, Cancer Res48:2231-2238; Yamazaki et al., 1990, supra). EGFRvIII has been shown tohave a ligand-independent, weak but constitutively active kinaseactivity as well as enhanced tumorigenicity (Nishikawa et al., 1994,Proc Natl Acad Sci USA 91:7727-7731; and Batra et al., 1995, Cell Growthand Differentiation 6:1251-1259). In addition to gliomas, EGFRvIII hasbeen detected in ductal and intraductal breast carcinoma (Wikstrand etal., 1995, Cancer Res 55:3140-3148), non-small cell lung carcinomas(Garcia de Palazzo et al., 1993, Cancer Res 53:3217-3220), ovariancarcinomas (Moscatello et al., 1995, Cancer Res 55:5536-5539), prostatecancer (Olapade-Olaopa et al., 2000, British J. Cancer 82:186-194), andsquamous cell carcinoma of the head and neck (Tinhofer et al., 2011,Clin Cancer Res 17(15):5197-5204). In contrast, these and other studiesreport that normal tissues do not express EGFRvIII (Garcia de Palazzo etal., 1993, supra; Wikstrand et al., 1995, supra; and Wikstrand et al.,1998, J Neuro Virol 4:148-158). The highly tumor-specific nature ofEGFRvIII makes it an especially useful target for treating cancers andtumors that express this molecule.

The nucleic acid and amino acid sequences of human EGFR are shown in SEQID NOs: 145 and 146, respectively, and the amino acid sequence ofEGFRvIII is shown in SEQ ID NO:147. Antibodies to EGFRvIII are describedin, for example, U.S. Pat. No. 5,212,290, U.S. Pat. No. 7,736,644, U.S.Pat. No. 7,589,180 and U.S. Pat. No. 7,767,792.

BRIEF SUMMARY OF THE INVENTION

The present invention provides antibodies and antigen-binding fragmentsthereof that bind EGFRvIII. The antibodies of the invention are useful,inter alia, for targeting tumor cells that express EGFRvIII. Theanti-EGFRvIII antibodies of the invention, and antigen-binding portionsthereof, may be used alone in unmodified form, or may be included aspart of an antibody-drug conjugate or a bispecific antibody.

The antibodies of the invention can be full-length (for example, an IgG1or IgG4 antibody) or may comprise only an antigen-binding portion (forexample, a Fab, F(ab′)₂ or scFv fragment), and may be modified to affectfunctionality, e.g., to eliminate residual effector functions (Reddy etal., 2000, J. Immunol. 164:1925-1933).

Exemplary anti-EGFRvIII antibodies of the present invention are listedin Tables 1 and 2 herein. Table 1 sets forth the amino acid sequenceidentifiers of the heavy chain variable regions (HCVRs), light chainvariable regions (LCVRs), heavy chain complementarity determiningregions (HCDR1, HCDR2 and HCDR3), and light chain complementaritydetermining regions (LCDR1, LCDR2 and LCDR3) of the exemplaryanti-EGFRvIII antibodies. Table 2 sets forth the nucleic acid sequenceidentifiers of the HCVRs, LCVRs, HCDR1, HCDR2 HCDR3, LCDR1, LCDR2 andLCDR3 of the exemplary anti-EGFRvIII antibodies.

The present invention provides antibodies or antigen-binding fragmentsthereof that specifically bind EGFRvIII, comprising an HCVR comprisingan amino acid sequence selected from any of the HCVR amino acidsequences listed in Table 1, or a substantially similar sequence thereofhaving at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity thereto.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind EGFRvIII, comprising an LCVRcomprising an amino acid sequence selected from any of the LCVR aminoacid sequences listed in Table 1, or a substantially similar sequencethereof having at least 90%, at least 95%, at least 98% or at least 99%sequence identity thereto.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind EGFRvIII, comprising an HCVRand an LCVR amino acid sequence pair (HCVR/LCVR) comprising any of theHCVR amino acid sequences listed in Table 1 paired with any of the LCVRamino acid sequences listed in Table 1. According to certainembodiments, the present invention provides antibodies, orantigen-binding fragments thereof, comprising an HCVR/LCVR amino acidsequence pair contained within any of the exemplary anti-EGFRvIIIantibodies listed in Table 1. In certain embodiments, the HCVR/LCVRamino acid sequence pair is selected from the group consisting of: 2/20,18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, and 130/138.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind EGFRvIII, comprising a heavychain CDR1 (HCDR1) comprising an amino acid sequence selected from anyof the HCDR1 amino acid sequences listed in Table 1 or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind EGFRvIII, comprising a heavychain CDR2 (HCDR2) comprising an amino acid sequence selected from anyof the HCDR2 amino acid sequences listed in Table 1 or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind EGFRvIII, comprising a heavychain CDR3 (HCDR3) comprising an amino acid sequence selected from anyof the HCDR3 amino acid sequences listed in Table 1 or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind EGFRvIII, comprising a lightchain CDR1 (LCDR1) comprising an amino acid sequence selected from anyof the LCDR1 amino acid sequences listed in Table 1 or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind EGFRvIII, comprising a lightchain CDR2 (LCDR2) comprising an amino acid sequence selected from anyof the LCDR2 amino acid sequences listed in Table 1 or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind EGFRvIII, comprising a lightchain CDR3 (LCDR3) comprising an amino acid sequence selected from anyof the LCDR3 amino acid sequences listed in Table 1 or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind EGFRvIII, comprising an HCDR3and an LCDR3 amino acid sequence pair (HCDR3/LCDR3) comprising any ofthe HCDR3 amino acid sequences listed in Table 1 paired with any of theLCDR3 amino acid sequences listed in Table 1. According to certainembodiments, the present invention provides antibodies, orantigen-binding fragments thereof, comprising an HCDR3/LCDR3 amino acidsequence pair contained within any of the exemplary anti-EGFRvIIIantibodies listed in Table 1.

The present invention also provides antibodies or antigen-bindingfragments thereof that specifically bind EGFRvIII, comprising a set ofsix CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained withinany of the exemplary anti-EGFRvIII antibodies listed in Table 1. Incertain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acidsequences set is selected from the group consisting of: 4-6-8-12-14-16;20-22-24-28-30-32; 36-38-40-44-46-48; 52-54-56-60-62-64;68-70-72-76-78-80; 84-86-88-92-94-96; 100-102-104-108-110-112;116-118-120-124-126-128; and 132-134-136-140-142-144.

In a related embodiment, the present invention provides antibodies, orantigen-binding fragments thereof that specifically bind EGFRvIII,comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3)contained within an HCVR/LCVR amino acid sequence pair as defined by anyof the exemplary anti-EGFRvIII antibodies listed in Table 1. Forexample, the present invention includes antibodies or antigen-bindingfragments thereof that specifically bind EGFRvIII, comprising theHCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences set containedwithin an HCVR/LCVR amino acid sequence pair selected from the groupconsisting of: 18/26; 66/74; 274/282; 290/298; and 370/378. Methods andtechniques for identifying CDRs within HCVR and LCVR amino acidsequences are well known in the art and can be used to identify CDRswithin the specified HCVR and/or LCVR amino acid sequences disclosedherein. Exemplary conventions that can be used to identify theboundaries of CDRs include, e.g., the Kabat definition, the Chothiadefinition, and the AbM definition. In general terms, the Kabatdefinition is based on sequence variability, the Chothia definition isbased on the location of the structural loop regions, and the AbMdefinition is a compromise between the Kabat and Chothia approaches.See, e.g., Kabat, “Sequences of Proteins of Immunological Interest,”National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al.,J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad.Sci. USA 86:9268-9272 (1989). Public databases are also available foridentifying CDR sequences within an antibody.

The present invention also provides nucleic acid molecules encodinganti-EGFRvIII antibodies or portions thereof. For example, the presentinvention provides nucleic acid molecules encoding any of the HCVR aminoacid sequences listed in Table 1; in certain embodiments the nucleicacid molecule comprises a polynucleotide sequence selected from any ofthe HCVR nucleic acid sequences listed in Table 2, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity thereto.

The present invention also provides nucleic acid molecules encoding anyof the LCVR amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCVR nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR1 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR2 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the HCDR3 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the HCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR1 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR1 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR2 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR2 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anyof the LCDR3 amino acid sequences listed in Table 1; in certainembodiments the nucleic acid molecule comprises a polynucleotidesequence selected from any of the LCDR3 nucleic acid sequences listed inTable 2, or a substantially similar sequence thereof having at least90%, at least 95%, at least 98% or at least 99% sequence identitythereto.

The present invention also provides nucleic acid molecules encoding anHCVR, wherein the HCVR comprises a set of three CDRs (i.e.,HCDR1-HCDR2-HCDR3), wherein the HCDR1-HCDR2-HCDR3 amino acid sequenceset is as defined by any of the exemplary anti-EGFRvIII antibodieslisted in Table 1.

The present invention also provides nucleic acid molecules encoding anLCVR, wherein the LCVR comprises a set of three CDRs (i.e.,LCDR1-LCDR2-LCDR3), wherein the LCDR1-LCDR2-LCDR3 amino acid sequenceset is as defined by any of the exemplary anti-EGFRvIII antibodieslisted in Table 1.

The present invention also provides nucleic acid molecules encoding bothan HCVR and an LCVR, wherein the HCVR comprises an amino acid sequenceof any of the HCVR amino acid sequences listed in Table 1, and whereinthe LCVR comprises an amino acid sequence of any of the LCVR amino acidsequences listed in Table 1. In certain embodiments, the nucleic acidmolecule comprises a polynucleotide sequence selected from any of theHCVR nucleic acid sequences listed in Table 2, or a substantiallysimilar sequence thereof having at least 90%, at least 95%, at least 98%or at least 99% sequence identity thereto, and a polynucleotide sequenceselected from any of the LCVR nucleic acid sequences listed in Table 2,or a substantially similar sequence thereof having at least 90%, atleast 95%, at least 98% or at least 99% sequence identity thereto. Incertain embodiments according to this aspect of the invention, thenucleic acid molecule encodes an HCVR and LCVR, wherein the HCVR andLCVR are both derived from the same anti-EGFRvIII antibody listed inTable 1.

The present invention also provides recombinant expression vectorscapable of expressing a polypeptide comprising a heavy or light chainvariable region of an anti-EGFRvIII antibody. For example, the presentinvention includes recombinant expression vectors comprising any of thenucleic acid molecules mentioned above, i.e., nucleic acid moleculesencoding any of the HCVR, LCVR, and/or CDR sequences as set forth inTable 1. Also included within the scope of the present invention arehost cells into which such vectors have been introduced, as well asmethods of producing the antibodies or portions thereof by culturing thehost cells under conditions permitting production of the antibodies orantibody fragments, and recovering the antibodies and antibody fragmentsso produced.

The present invention includes anti-EGFRvIII antibodies having amodified glycosylation pattern. In some embodiments, modification toremove undesirable glycosylation sites may be useful, or an antibodylacking a fucose moiety present on the oligosaccharide chain, forexample, to increase antibody dependent cellular cytotoxicity (ADCC)function (see Shield et al. (2002) JBC 277:26733). In otherapplications, modification of galactosylation can be made in order tomodify complement dependent cytotoxicity (CDC).

In another aspect, the invention provides a pharmaceutical compositioncomprising a recombinant human antibody or fragment thereof whichspecifically binds EGFRvIII and a pharmaceutically acceptable carrier.In a related aspect, the invention features a composition which is acombination of an anti-EGFRvIII antibody and a second therapeutic agent.In one embodiment, the second therapeutic agent is any agent that isadvantageously combined with an anti-EGFRvIII antibody. The presentinvention also provides antibody-drug conjugates (ADCs) comprising ananti-EGFRvIII antibody conjugated to a cytotoxic agent. Exemplarycombination therapies, co-formulations, and ADCs involving theanti-EGFRvIII antibodies of the present invention are disclosedelsewhere herein.

In yet another aspect, the invention provides therapeutic methods forkilling tumor cells or for inhibiting or attenuating tumor cell growthusing an anti-EGFRvIII antibody or antigen-binding portion of anantibody of the invention. The therapeutic methods according to thisaspect of the invention comprise administering a therapeuticallyeffective amount of a pharmaceutical composition comprising an antibodyor antigen-binding fragment of an antibody of the invention to a subjectin need thereof. The disorder treated is any disease or condition whichis improved, ameliorated, inhibited or prevented by targeting EGFRvIIIand/or by inhibiting ligand-mediated cell signaling through EGFRvIII.

Other embodiments will become apparent from a review of the ensuingdetailed description. Other embodiments will become apparent from areview of the ensuing detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1(a-b) shows the results of western blot of EGFR and EGFRvIII usinganti-EGFRvIII antibodies [i.e., H1H1863N2(Fuc−), and Controls I and IIin FIG. 1a ; and H1H1911, H1H1912, and H1H1915 in FIG. 1b ], or anti-Hisantibody, under reduced (upper panels) and non-reduced (lower panels)conditions. Lanes 1 and 6: 10 μl of BENCHMARK™ standard (INVITROGEN™);Lanes 2 and 7: 400 ng of hEGFR-mmh (SEQ ID NO:154); Lane 3 and 8: 400 ngof hEGFRvIII-mmh (SEQ ID NO:152); and Lanes 4, 5, 9 and 10: space.Control I: Human anti-EGFRvIII junctional peptide antibody (IgG1)disclosed in U.S. Pat. No. 7,736,644; and Control II: Chimericanti-EGFRvIII/EGFR antibody disclosed in U.S. Pat. No. 7,589,180.

FIG. 2 shows the binding characteristics of H1H1863N2(Fuc−). TheEGFRvIII junctional peptide or the peptide of residues 311-326 of EGFR(“EGFR311-326 peptide”), each of which was tagged via a linker withbiotin at the C-terminus, was captured to streptavidin-coated OCTET®tips on a FORTEBIO® OCTET® RED instrument and reacted withH1H1863N2(Fuc−) or Control I-III. Controls I and II: Same as above; andControl III: Humanized anti-EGFRvIII antibody (hIgG1) disclosed in USPatent Application Publication No. 2010/0056762. (□): C-terminalbiotin-labeled EGFRvIII junctional peptide (SEQ ID NO:149); and (▪):C-terminal biotin-labeled EGFR311-326 peptide (SEQ ID NO:151).

FIG. 3 shows the internalization of anti-EGFRvIII mAb by HEK293 cellsexpressing EGFRvIII (HEK293/EGFRvIII). Cell-surface bound anti-EGFRvIIIantibodies and control antibodies were detected by dye-conjugatedsecondary antibody (Fab); images were acquired at 40× and internalizedvesicles were quantitated. Controls I and II: Same as above; and ControlIV: Chimeric anti-EGFR antibody disclosed in U.S. Pat. No. 7,060,808.(□): Internalization at 37° C.; and (▪): Internalization at 4° C.

FIG. 4(a-b) shows the binding and internalization of anti-EGFRvIIIantibody H1H1863N2(Fuc−) by B16F10.9 tumors or B16F10.9 tumorsexpressing EGFRvIII (B16F10.9/EGFRvIII) that were xenografted in severecombined immunodeficient (SCID) mice. Cell-surface bound (FIG. 4a ) orcell-surface-bound plus internalized (FIG. 4b ) anti-EGFRvIII antibodyor isotype control antibody, was detected by allophycocyanin conjugatedanti-human Fc (hFc-APC) antibody using flow cytometry. Mean fluorescentintensities (MIF) at 10 minutes (▪), 4 hours (

), and 24 hours (□), post-antibody injection, are shown.

FIG. 5(a-d) shows the results of pharmacokinetics analysis foranti-EGFRvIII antibody H1H863N2(Fuc+) (FIG. 5d ) and control antibodies(as described above), i.e., Control I (FIG. 5b ), Control III (FIG. 5c), and Control IV (FIG. 5a ), in wild-type mice (●) or mice expressinghuman EGFR (▪).

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

Definitions

The term “EGFRvIII,” as used herein, refers to the human EGFR class IIIvariant having the amino acid sequence shown in SEQ ID NO:147, or abiologically active fragment thereof, which exhibits any characteristicsspecific for EGFRvIII, as opposed to those in common with normallyexpressed EGFR, unless specifically indicated otherwise. EGFRvIII lacksamino acid residues 6 through 273 of mature EGFR (i.e., SEQ ID NO:146without the signal peptide, i.e., residues 1-24) and contains a newglycine residue at position 6 between amino acid residues 5 and 274.

All references to proteins, polypeptides and protein fragments hereinare intended to refer to the human version of the respective protein,polypeptide or protein fragment unless explicitly specified as beingfrom a non-human species. Thus, the expression “EGFRvIII” means humanEGFRvIII unless specified as being from a non-human species, e.g.,“mouse EGFRvIII,” “monkey EGFRvIII,” etc.

As used herein, the expression “cell surface-expressed EGFRvIII” meansone or more EGFRvIII protein(s), or the extracellular domain thereof,that is/are expressed on the surface of a cell in vitro or in vivo, suchthat at least a portion of a EGFRvIII protein is exposed to theextracellular side of the cell membrane and is accessible to anantigen-binding portion of an antibody. A “cell surface-expressedEGFRvIII” can comprise or consist of an EGFRvIII protein expressed onthe surface of a cell which normally expresses EGFRvIII protein.Alternatively, “cell surface-expressed EGFRvIII” can comprise or consistof EGFRvIII protein expressed on the surface of a cell that normallydoes not express human EGFRvIII on its surface but has been artificiallyengineered to express EGFRvIII on its surface.

As used herein, the expression “anti-EGFRvIII antibody” includes bothmonovalent antibodies with a single specificity, as well as bispecificantibodies comprising a first arm that binds EGFRvIII and a second armthat binds a second (target) antigen, wherein the anti-EGFRvIII armcomprises any of the HCVR/LCVR or CDR sequences as set forth in Table 1herein. The expression “anti-EGFRvIII antibody” also includesantibody-drug conjugates (ADCs) comprising an anti-EGFRvIII antibody orantigen-binding portion thereof conjugated to a drug or toxin (i.e.,cytotoxic agent). The expression “anti-EGFRvIII antibody” also includesantibody-radionuclide conjugates (ARCs) comprising an anti-EGFRvIIIantibody or antigen-binding portion thereof conjugated to aradionuclide.

The term “antibody”, as used herein, means any antigen-binding moleculeor molecular complex comprising at least one complementarity determiningregion (CDR) that specifically binds to or interacts with a particularantigen (e.g., EGFRvIII). The term “antibody” includes immunoglobulinmolecules comprising four polypeptide chains, two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds, as well asmultimers thereof (e.g., IgM). Each heavy chain comprises a heavy chainvariable region (abbreviated herein as HCVR or V_(H)) and a heavy chainconstant region. The heavy chain constant region comprises threedomains, C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises a lightchain variable region (abbreviated herein as LCVR or V_(L)) and a lightchain constant region. The light chain constant region comprises onedomain (C_(L)1). The V_(H) and V_(L) regions can be further subdividedinto regions of hypervariability, termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FR). Each V_(H) and V_(L) is composed of threeCDRs and four FRs, arranged from amino-terminus to carboxy-terminus inthe following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In differentembodiments of the invention, the FRs of the anti-EGFRvIII antibody (orantigen-binding portion thereof) may be identical to the human germlinesequences, or may be naturally or artificially modified. An amino acidconsensus sequence may be defined based on a side-by-side analysis oftwo or more CDRs.

The term “antibody”, as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in noncovalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

The antibodies of the present invention may function throughcomplement-dependent cytotoxicity (CDC) or antibody-dependentcell-mediated cytotoxicity (ADCC). “Complement-dependent cytotoxicity”(CDC) refers to lysis of antigen-expressing cells by an antibody of theinvention in the presence of complement. “Antibody-dependentcell-mediated cytotoxicity” (ADCC) refers to a cell-mediated reaction inwhich nonspecific cytotoxic cells that express Fc receptors (FcRs)(e.g., Natural Killer (NK) cells, neutrophils, and macrophages)recognize bound antibody on a target cell and thereby lead to lysis ofthe target cell. CDC and ADCC can be measured using assays that are wellknown and available in the art. (See, e.g., U.S. Pat. Nos. 5,500,362 and5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA)95:652-656). The constant region of an antibody is important in theability of an antibody to fix complement and mediate cell-dependentcytotoxicity. Thus, the isotype of an antibody may be selected on thebasis of whether it is desirable for the antibody to mediatecytotoxicity.

In certain embodiments of the invention, the anti-EGFRvIII antibodies ofthe invention are human antibodies. The term “human antibody”, as usedherein, is intended to include antibodies having variable and constantregions derived from human germline immunoglobulin sequences. The humanantibodies of the invention may include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo), for example in the CDRs and in particular CDR3. However, theterm “human antibody”, as used herein, is not intended to includeantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto humanframework sequences.

The antibodies of the invention may, in some embodiments, be recombinanthuman antibodies. The term “recombinant human antibody”, as used herein,is intended to include all human antibodies that are prepared,expressed, created or isolated by recombinant means, such as antibodiesexpressed using a recombinant expression vector transfected into a hostcell (described further below), antibodies isolated from a recombinant,combinatorial human antibody library (described further below),antibodies isolated from an animal (e.g., a mouse) that is transgenicfor human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl.Acids Res. 20:6287-6295) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the V_(H) andV_(L) regions of the recombinant antibodies are sequences that, whilederived from and related to human germline V_(H) and V_(L) sequences,may not naturally exist within the human antibody germline repertoire invivo.

Human antibodies can exist in two forms that are associated with hingeheterogeneity. In one form, an immunoglobulin molecule comprises astable four chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via interchain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a covalentlycoupled light and heavy chain (half-antibody). These forms have beenextremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et al.(1993) Molecular Immunology 30:105) to levels typically observed using ahuman IgG1 hinge. The instant invention encompasses antibodies havingone or more mutations in the hinge, C_(H)2 or C_(H)3 region which may bedesirable, for example, in production, to improve the yield of thedesired antibody form.

The antibodies of the invention may be isolated antibodies. An “isolatedantibody,” as used herein, means an antibody that has been identifiedand separated and/or recovered from at least one component of itsnatural environment. For example, an antibody that has been separated orremoved from at least one component of an organism, or from a tissue orcell in which the antibody naturally exists or is naturally produced, isan “isolated antibody” for purposes of the present invention. Anisolated antibody also includes an antibody in situ within a recombinantcell. Isolated antibodies are antibodies that have been subjected to atleast one purification or isolation step. According to certainembodiments, an isolated antibody may be substantially free of othercellular material and/or chemicals.

The anti-EGFRvIII antibodies disclosed herein may comprise one or moreamino acid substitutions, insertions and/or deletions in the frameworkand/or CDR regions of the heavy and light chain variable domains ascompared to the corresponding germline sequences from which theantibodies were derived. Such mutations can be readily ascertained bycomparing the amino acid sequences disclosed herein to germlinesequences available from, for example, public antibody sequencedatabases. The present invention includes antibodies, andantigen-binding fragments thereof, which are derived from any of theamino acid sequences disclosed herein, wherein one or more amino acidswithin one or more framework and/or CDR regions are mutated to thecorresponding residue(s) of the germline sequence from which theantibody was derived, or to the corresponding residue(s) of anotherhuman germline sequence, or to a conservative amino acid substitution ofthe corresponding germline residue(s) (such sequence changes arereferred to herein collectively as “germline mutations”). A person ofordinary skill in the art, starting with the heavy and light chainvariable region sequences disclosed herein, can easily produce numerousantibodies and antigen-binding fragments which comprise one or moreindividual germline mutations or combinations thereof. In certainembodiments, all of the framework and/or CDR residues within the V_(H)and/or V_(L) domains are mutated back to the residues found in theoriginal germline sequence from which the antibody was derived. In otherembodiments, only certain residues are mutated back to the originalgermline sequence, e.g., only the mutated residues found within thefirst 8 amino acids of FR1 or within the last 8 amino acids of FR4, oronly the mutated residues found within CDR1, CDR2 or CDR3. In otherembodiments, one or more of the framework and/or CDR residue(s) aremutated to the corresponding residue(s) of a different germline sequence(i.e., a germline sequence that is different from the germline sequencefrom which the antibody was originally derived). Furthermore, theantibodies of the present invention may contain any combination of twoor more germline mutations within the framework and/or CDR regions,e.g., wherein certain individual residues are mutated to thecorresponding residue of a particular germline sequence while certainother residues that differ from the original germline sequence aremaintained or are mutated to the corresponding residue of a differentgermline sequence. Once obtained, antibodies and antigen-bindingfragments that contain one or more germline mutations can be easilytested for one or more desired property such as, improved bindingspecificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. Antibodies and antigen-binding fragmentsobtained in this general manner are encompassed within the presentinvention.

The present invention also includes anti-EGFRvIII antibodies comprisingvariants of any of the HCVR, LCVR, and/or CDR amino acid sequencesdisclosed herein having one or more conservative substitutions. Forexample, the present invention includes anti-EGFRvIII antibodies havingHCVR, LCVR, and/or CDR amino acid sequences with, e.g., 10 or fewer, 8or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acidsubstitutions relative to any of the HCVR, LCVR, and/or CDR amino acidsequences set forth in Table 1 herein.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95%, and more preferablyat least about 96%, 97%, 98% or 99% of the nucleotide bases, as measuredby any well-known algorithm of sequence identity, such as FASTA, BLASTor Gap, as discussed below. A nucleic acid molecule having substantialidentity to a reference nucleic acid molecule may, in certain instances,encode a polypeptide having the same or substantially similar amino acidsequence as the polypeptide encoded by the reference nucleic acidmolecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 95% sequence identity, even more preferably atleast 98% or 99% sequence identity. Preferably, residue positions whichare not identical differ by conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See, e.g., Pearson (1994)Methods Mol. Biol. 24: 307-331, herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include (1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains:serine and threonine; (3) amide-containing side chains: asparagine andglutamine; (4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; (5) basic side chains: lysine, arginine, and histidine; (6)acidic side chains: aspartate and glutamate, and (7) sulfur-containingside chains are cysteine and methionine. Preferred conservative aminoacids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443-1445, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389-402, each herein incorporated byreference.

pH-Dependent Binding

The present invention includes anti-EGFRvIII antibodies withpH-dependent binding characteristics. For example, an anti-EGFRvIIIantibody of the present invention may exhibit reduced binding toEGFRvIII at acidic pH as compared to neutral pH. Alternatively,anti-EGFRvIII antibodies of the invention may exhibit enhanced bindingto EGFRvIII at acidic pH as compared to neutral pH. The expression“acidic pH” includes pH values less than about 6.2, e.g., about 6.0,5.95, 5, 9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35,5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, or less. As used herein, theexpression “neutral pH” means a pH of about 7.0 to about 7.4. Theexpression “neutral pH” includes pH values of about 7.0, 7.05, 7.1,7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

In certain instances, “reduced binding to EGFRvIII at acidic pH ascompared to neutral pH” is expressed in terms of a ratio of the K_(D)value of the antibody binding to EGFRvIII at acidic pH to the K_(D)value of the antibody binding to EGFRvIII at neutral pH (or vice versa).For example, an antibody or antigen-binding fragment thereof may beregarded as exhibiting “reduced binding to EGFRvIII at acidic pH ascompared to neutral pH” for purposes of the present invention if theantibody or antigen-binding fragment thereof exhibits an acidic/neutralK_(D) ratio of about 3.0 or greater. In certain exemplary embodiments,the acidic/neutral K_(D) ratio for an antibody or antigen-bindingfragment of the present invention can be about 3.0, 3.5, 4.0, 4.5, 5.0,5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5,12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0. 25.0, 30.0, 40.0, 50.0,60.0, 70.0, 100.0 or greater.

Antibodies with pH-dependent binding characteristics may be obtained,e.g., by screening a population of antibodies for reduced (or enhanced)binding to a particular antigen at acidic pH as compared to neutral pH.Additionally, modifications of the antigen-binding domain at the aminoacid level may yield antibodies with pH-dependent characteristics. Forexample, by substituting one or more amino acids of an antigen-bindingdomain (e.g., within a CDR) with a histidine residue, an antibody withreduced antigen-binding at acidic pH relative to neutral pH may beobtained.

Anti-EGFRvIII Antibodies Comprising Fc Variants

According to certain embodiments of the present invention, anti-EGFRvIIIantibodies are provided comprising an Fc domain comprising one or moremutations which enhance or diminish antibody binding to the FcRnreceptor, e.g., at acidic pH as compared to neutral pH. For example, thepresent invention includes anti-EGFRvIII antibodies comprising amutation in the C_(H)2 or a C_(H)3 region of the Fc domain, wherein themutation(s) increases the affinity of the Fc domain to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0). Such mutations may result in an increase in serum half-lifeof the antibody when administered to an animal. Non-limiting examples ofsuch Fc modifications include, e.g., a modification at position 250(e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T),254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification atposition 428 and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., A, W,H, F or Y [N434A, N434W, N434H, N434F or N434Y]); or a modification atposition 250 and/or 428; or a modification at position 307 or 308 (e.g.,308F, V308F), and 434. In one embodiment, the modification comprises a428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I(e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K)and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y,254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Qand M428L); and a 307 and/or 308 modification (e.g., 308F or 308P). Inyet another embodiment, the modification comprises a 265A (e.g., D265A)and/or a 297A (e.g., N297A) modification.

For example, the present invention includes anti-EGFRvIII antibodiescomprising an Fc domain comprising one or more pairs or groups ofmutations selected from the group consisting of: 250Q and 248L (e.g.,T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E);428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I andQ311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g.,D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A);and 433K and 434F (e.g., H433K and N434F). All possible combinations ofthe foregoing Fc domain mutations, and other mutations within theantibody variable domains disclosed herein, are contemplated within thescope of the present invention.

The present invention also includes anti-EGFRvIII antibodies comprisinga chimeric heavy chain constant (C_(H)) region, wherein the chimericC_(H) region comprises segments derived from the C_(H) regions of morethan one immunoglobulin isotype. For example, the antibodies of theinvention may comprise a chimeric C_(H) region comprising part or all ofa C_(H)2 domain derived from a human IgG1, human IgG2 or human IgG4molecule, combined with part or all of a C_(H)3 domain derived from ahuman IgG1, human IgG2 or human IgG4 molecule. According to certainembodiments, the antibodies of the invention comprise a chimeric C_(H)region having a chimeric hinge region. For example, a chimeric hinge maycomprise an “upper hinge” amino acid sequence (amino acid residues frompositions 216 to 227 according to EU numbering) derived from a humanIgG1, a human IgG2 or a human IgG4 hinge region, combined with a “lowerhinge” sequence (amino acid residues from positions 228 to 236 accordingto EU numbering) derived from a human IgG1, a human IgG2 or a human IgG4hinge region. According to certain embodiments, the chimeric hingeregion comprises amino acid residues derived from a human IgG1 or ahuman IgG4 upper hinge and amino acid residues derived from a human IgG2lower hinge. An antibody comprising a chimeric C_(H) region as describedherein may, in certain embodiments, exhibit modified Fc effectorfunctions without adversely affecting the therapeutic or pharmacokineticproperties of the antibody. (See, e.g., U.S. Provisional Appl. No.61/759,578, filed Feb. 1, 2013, the disclosure of which is herebyincorporated by reference in its entirety).

Antibody-Drug Conjugates (ADCs)

The present invention provides antibody-drug conjugates (ADCs)comprising an anti-EGFRvIII antibody or antigen-binding fragment thereofconjugated to a therapeutic moiety such as a cytotoxic agent, achemotherapeutic drug, or a radioisotope.

Cytotoxic agents include any agent that is detrimental to the growth,viability or propagation of cells. Examples of suitable cytotoxic agentsand chemotherapeutic agents that can be conjugated to anti-EGFRvIIIantibodies in accordance with this aspect of the invention include,e.g., 1-(2chloroethyl)-1,2-dimethanesulfonyl hydrazide,1,8-dihydroxy-bicyclo[7.3.1]trideca-4,9-diene-2,6-diyne-13-one,1-dehydrotestosterone, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine,9-amino camptothecin, actinomycin D, amanitins, aminopterin, anguidine,anthracycline, anthramycin (AMC), auristatins, bleomycin, busulfan,butyric acid, calicheamicins, camptothecin, carminomycins, carmustine,cemadotins, cisplatin, colchicin, combretastatins, cyclophosphamide,cytarabine, cytochalasin B, dactinomycin, daunorubicin, decarbazine,diacetoxypentyldoxorubicin, dibromomannitol, dihydroxy anthracin dione,disorazoles, dolastatin, doxorubicin, duocarmycin, echinomycins,eleutherobins, emetine, epothilones, esperamicin, estramustines,ethidium bromide, etoposide, fluorouracils, geldanamycins, gramicidin D,glucocorticoids, irinotecans, leptomycins, leurosines, lidocaine,lomustine (CCNU), maytansinoids, mechlorethamine, melphalan,mercatopurines, methopterins, methotrexate, mithramycin, mitomycin,mitoxantrone, N8-acetyl spermidine, podophyllotoxins, procaine,propranolol, pteridines, puromycin, pyrrolobenzodiazepines (PDBs),rhizoxins, streptozotocin, tallysomycins, taxol, tenoposide, tetracaine,thioepa chlorambucil, tomaymycins, topotecans, tubulysin, vinblastine,vincristine, vindesine, vinorelbines, and derivatives of any of theforegoing. According to certain embodiments, the cytotoxic agent that isconjugated to an anti-EGFRvIII antibody is a maytansinoid such as DM1 orDM4, a tomaymycin derivative, or a dolastatin derivative. Othercytotoxic agents known in the art are contemplated within the scope ofthe present invention, including, e.g., protein toxins such ricin, C.difficile toxin, pseudomonas exotoxin, ricin, diphtheria toxin,botulinum toxin, bryodin, saporin, pokeweed toxins (i.e.,phytolaccatoxin and phytolaccigenin), and others such as those set forthin Sapra et al., Pharmacol. & Therapeutics, 2013, 138:452-469.

The present invention also includes antibody-radionuclide conjugates(ARCs) comprising anti-EGFRvIII antibodies conjugated to one or moreradionuclides. Exemplary radionuclides that can be used in the contextof this aspect of the invention include, but are not limited to, e.g.,²²⁵Ac, ²¹²Bi, ²¹³Bi, ¹³¹I, ¹⁸⁶Re, ²²⁷Th, ²²²Rn, ²²³Ra, ²²⁴Ra, and ⁹⁰Y.

In certain embodiments of the present invention, ADCs are providedcomprising an anti-EGFRvIII antibody conjugated to a cytotoxic agent(e.g., any of the cytotoxic agents disclosed above) via a linkermolecule. Any linker molecule or linker technology known in the art canbe used to create or construct an ADC of the present invention. Incertain embodiments, the linker is a cleavable linker. According toother embodiments, the linker is a non-cleavable linker. Exemplarylinkers that can be used in the context of the present inventioninclude, linkers that comprise or consist of e.g., MC(6-maleimidocaproyl), MP (maleimidopropanoyl), val-cit(valine-citrulline), val-ala (valine-alanine), dipeptide site inprotease-cleavable linker, ala-phe (alanine-phenylalanine), dipeptidesite in protease-cleavable linker, PAB (p-aminobenzyloxycarbonyl), SPP(N-Succinimidyl 4-(2-pyridylthio) pentanoate), SMCC (N-Succinimidyl4-(N-maleimidomethyl)cyclohexane-1 carboxylate), STAB (N-Succinimidyl(4-iodo-acetyl)aminobenzoate), and variants and combinations thereof.Additional examples of linkers that can be used in the context of thepresent invention are disclosed, e.g., in U.S. Pat. No. 7,754,681 and inDucry, Bioconjugate Chem., 2010, 21:5-13, and the references citedtherein, the contents of which are incorporated by reference herein intheir entireties.

The present invention comprises ADCs in which a linker connects ananti-EGFRvIII antibody or antigen-binding molecule to a drug orcytotoxin through an attachment at a particular amino acid within theantibody or antigen-binding molecule. Exemplary amino acid attachmentsthat can be used in the context of this aspect of the invention include,e.g., lysine (see, e.g., U.S. Pat. No. 5,208,020; US 2010/0129314;Hollander et al., Bioconjugate Chem., 2008, 19:358-361; WO 2005/089808;U.S. Pat. No. 5,714,586; US 2013/0101546; and US 2012/0585592), cysteine(see, e.g., US 2007/0258987; WO 2013/055993; WO 2013/055990; WO2013/053873; WO 2013/053872; WO 2011/130598; US 2013/0101546; and U.S.Pat. No. 7,750,116), selenocysteine (see, e.g., WO 2008/122039; andHofer et al., Proc. Natl. Acad. Sci., USA, 2008, 105:12451-12456),formyl glycine (see, e.g., Carrico et al., Nat. Chem. Biol., 2007,3:321-322; Agarwal et al., Proc. Natl. Acad. Sci., USA, 2013, 110:46-51,and Rabuka et al., Nat. Protocols, 2012, 10:1052-1067), non-naturalamino acids (see, e.g., WO 2013/068874, and WO 2012/166559), and acidicamino acids (see, e.g., WO 2012/05982). Linkers can also be conjugatedto an antigen-binding protein via attachment to carbohydrates (see,e.g., US 2008/0305497, and Ryan et al., Food & Agriculture Immunol.,2001, 13:127-130) and disulfide linkers (see, e.g., WO 2013/085925, WO2010/010324, WO 2011/018611, and Shaunak et al., Nat. Chem. Biol., 2006,2:312-313).

Any method known in the art for conjugating a chemical moiety to apeptide, polypeptide or other macromolecule can be used in the contextof the present invention to make an anti-EGFRvIII ADC as describedherein. An exemplary method for antibody-drug conjugation via a linkeris set forth in Example 12 herein. Variations on this exemplary methodwill be appreciated by persons of ordinary skill in the art and arecontemplated within the scope of the present invention.

According to certain embodiments, the present invention provides ADCs,wherein an anti-EGFRvIII antibody as described herein (e.g., theantibody designated H1H1863N2) is conjugated to a linker-drugcomposition as set forth in WO2014/145090 (e.g., compound “7,” alsoreferred to herein as “M0026”), the disclosure of which is herebyincorporated by reference herein in its entirety (see also Example 12,herein).

Epitope Mapping and Related Technologies

The epitope to which the antibodies of the present invention bind mayconsist of a single contiguous sequence of 3 or more (e.g., 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acidsof an EGFRvIII protein. Alternatively, the epitope may consist of aplurality of non-contiguous amino acids (or amino acid sequences) ofEGFRvIII. In some embodiments, the epitope is located on or near theligand-binding domain of EGFRvIII. In other embodiments, the epitope islocated outside of the ligand-binding domain of EGFRvIII, e.g., at alocation on the surface of EGFRvIII at which an antibody, when bound tosuch an epitope, does not interfere with ligand binding to EGFRvIII.

The present invention, according to certain embodiments, includesanti-EGFRvIII antibodies that specifically bind EGFRvIII (and do notbind EGFR), wherein the antibodies recognize the EGFRvIII junctionalpeptide (e.g., SEQ ID NO:148). Such antibodies may be referred to hereinas “junctional peptide binders,” “EGFRvIII peptide-binding antibodies,”and the like. The present invention, according to other embodiments,includes anti-EGFRvIII antibodies that specifically bind EGFRvIII (anddo not bind EGFR), wherein the antibodies do not recognize the EGFRvIIIjunctional peptide (e.g. do not recognize the junctional peptide of SEQID NO:148, and/or do not recognize the peptide of SEQ ID NO:165). Suchantibodies may be referred to herein as “conformational binders,”“EGFRvIII conformational epitope binders,” and the like.

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antibody “interacts with one or more aminoacids” within a polypeptide or protein. Exemplary techniques include,e.g., routine cross-blocking assay such as that described Antibodies,Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., N.Y.),alanine scanning mutational analysis, peptide blots analysis (Reineke,2004, Methods Mol Biol 248:443-463), and peptide cleavage analysis. Inaddition, methods such as epitope excision, epitope extraction andchemical modification of antigens can be employed (Tomer, 2000, ProteinScience 9:487-496). Another method that can be used to identify theamino acids within a polypeptide with which an antibody interacts ishydrogen/deuterium exchange detected by mass spectrometry. In generalterms, the hydrogen/deuterium exchange method involvesdeuterium-labeling the protein of interest, followed by binding theantibody to the deuterium-labeled protein. Next, the protein/antibodycomplex is transferred to water to allow hydrogen-deuterium exchange tooccur at all residues except for the residues protected by the antibody(which remain deuterium-labeled). After dissociation of the antibody,the target protein is subjected to protease cleavage and massspectrometry analysis, thereby revealing the deuterium-labeled residueswhich correspond to the specific amino acids with which the antibodyinteracts. See, e.g., Ehring (1999) Analytical Biochemistry267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

The present invention further includes anti-EGFRvIII antibodies thatbind to the same epitope as any of the specific exemplary antibodiesdescribed herein (e.g. antibodies comprising any of the amino acidsequences as set forth in Table 1 herein). Likewise, the presentinvention also includes anti-EGFRvIII antibodies that compete forbinding to EGFRvIII with any of the specific exemplary antibodiesdescribed herein (e.g. antibodies comprising any of the amino acidsequences as set forth in Table 1 herein).

One can easily determine whether an antibody binds to the same epitopeas, or competes for binding with, a reference anti-EGFRvIII antibody byusing routine methods known in the art and exemplified herein. Forexample, to determine if a test antibody binds to the same epitope as areference anti-EGFRvIII antibody of the invention, the referenceantibody is allowed to bind to a EGFRvIII protein. Next, the ability ofa test antibody to bind to the EGFRvIII molecule is assessed. If thetest antibody is able to bind to EGFRvIII following saturation bindingwith the reference anti-EGFRvIII antibody, it can be concluded that thetest antibody binds to a different epitope than the referenceanti-EGFRvIII antibody. On the other hand, if the test antibody is notable to bind to the EGFRvIII molecule following saturation binding withthe reference anti-EGFRvIII antibody, then the test antibody may bind tothe same epitope as the epitope bound by the reference anti-EGFRvIIIantibody of the invention. Additional routine experimentation (e.g.,peptide mutation and binding analyses) can then be carried out toconfirm whether the observed lack of binding of the test antibody is infact due to binding to the same epitope as the reference antibody or ifsteric blocking (or another phenomenon) is responsible for the lack ofobserved binding. Experiments of this sort can be performed using ELISA,RIA, Biacore, flow cytometry or any other quantitative or qualitativeantibody-binding assay available in the art. In accordance with certainembodiments of the present invention, two antibodies bind to the same(or overlapping) epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excessof one antibody inhibits binding of the other by at least 50% butpreferably 75%, 90% or even 99% as measured in a competitive bindingassay (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502).Alternatively, two antibodies are deemed to bind to the same epitope ifessentially all amino acid mutations in the antigen that reduce oreliminate binding of one antibody reduce or eliminate binding of theother. Two antibodies are deemed to have “overlapping epitopes” if onlya subset of the amino acid mutations that reduce or eliminate binding ofone antibody reduce or eliminate binding of the other.

To determine if an antibody competes for binding (or cross-competes forbinding) with a reference anti-EGFRvIII antibody, the above-describedbinding methodology is performed in two orientations: In a firstorientation, the reference antibody is allowed to bind to an EGFRvIIIprotein under saturating conditions followed by assessment of binding ofthe test antibody to the EGFRvIII molecule. In a second orientation, thetest antibody is allowed to bind to an EGFRvIII molecule undersaturating conditions followed by assessment of binding of the referenceantibody to the EGFRvIII molecule. If, in both orientations, only thefirst (saturating) antibody is capable of binding to the EGFRvIIImolecule, then it is concluded that the test antibody and the referenceantibody compete for binding to EGFRvIII. As will be appreciated by aperson of ordinary skill in the art, an antibody that competes forbinding with a reference antibody may not necessarily bind to the sameepitope as the reference antibody, but may sterically block binding ofthe reference antibody by binding an overlapping or adjacent epitope.

Preparation of Human Antibodies

The anti-EGFRvIII antibodies of the present invention can be fully humanantibodies. Methods for generating monoclonal antibodies, includingfully human monoclonal antibodies are known in the art. Any such knownmethods can be used in the context of the present invention to makehuman antibodies that specifically bind to human EGFRvIII.

Using VELOCIMMUNE™ technology, for example, or any other similar knownmethod for generating fully human monoclonal antibodies, high affinitychimeric antibodies to EGFRvIII are initially isolated having a humanvariable region and a mouse constant region. As in the experimentalsection below, the antibodies are characterized and selected fordesirable characteristics, including affinity, ligand blocking activity,selectivity, epitope, etc. If necessary, mouse constant regions arereplaced with a desired human constant region, for example wild-type ormodified IgG1 or IgG4, to generate a fully human anti-EGFRvIII antibody.While the constant region selected may vary according to specific use,high affinity antigen-binding and target specificity characteristicsreside in the variable region. In certain instances, fully humananti-EGFRvIII antibodies are isolated directly from antigen-positive Bcells.

Bioequivalents

The anti-EGFRvIII antibodies and antibody fragments of the presentinvention encompass proteins having amino acid sequences that vary fromthose of the described antibodies but that retain the ability to bindhuman EGFRvIII. Such variant antibodies and antibody fragments compriseone or more additions, deletions, or substitutions of amino acids whencompared to parent sequence, but exhibit biological activity that isessentially equivalent to that of the described antibodies. Likewise,the anti-EGFRvIII antibody-encoding DNA sequences of the presentinvention encompass sequences that comprise one or more additions,deletions, or substitutions of nucleotides when compared to thedisclosed sequence, but that encode an anti-EGFRvIII antibody orantibody fragment that is essentially bioequivalent to an anti-EGFRvIIIantibody or antibody fragment of the invention. Examples of such variantamino acid and DNA sequences are discussed above.

Two antigen-binding proteins, or antibodies, are consideredbioequivalent if, for example, they are pharmaceutical equivalents orpharmaceutical alternatives whose rate and extent of absorption do notshow a significant difference when administered at the same molar doseunder similar experimental conditions, either single does or multipledose. Some antibodies will be considered equivalents or pharmaceuticalalternatives if they are equivalent in the extent of their absorptionbut not in their rate of absorption and yet may be consideredbioequivalent because such differences in the rate of absorption areintentional and are reflected in the labeling, are not essential to theattainment of effective body drug concentrations on, e.g., chronic use,and are considered medically insignificant for the particular drugproduct studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antibody.

Bioequivalent variants of anti-EGFRvIII antibodies of the invention maybe constructed by, for example, making various substitutions of residuesor sequences or deleting terminal or internal residues or sequences notneeded for biological activity. For example, cysteine residues notessential for biological activity can be deleted or replaced with otheramino acids to prevent formation of unnecessary or incorrectintramolecular disulfide bridges upon renaturation. In other contexts,bioequivalent antibodies may include anti-EGFRvIII antibody variantscomprising amino acid changes which modify the glycosylationcharacteristics of the antibodies, e.g., mutations which eliminate orremove glycosylation.

Species Selectivity and Species Cross-Reactivity

The present invention, according to certain embodiments, providesanti-EGFRvIII antibodies that bind to human EGFRvIII but not to EGFRvIIIfrom other species. The present invention also includes anti-EGFRvIIIantibodies that bind to human EGFRvIII and to EGFRvIII from one or morenon-human species. For example, the anti-EGFRvIII antibodies of theinvention may bind to human EGFRvIII and may bind or not bind, as thecase may be, to one or more of mouse, rat, guinea pig, hamster, gerbil,pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomologous,marmoset, rhesus or chimpanzee EGFRvIII. According to certain exemplaryembodiments of the present invention, anti-EGFRvIII antibodies areprovided which specifically bind human EGFRvIII and cynomolgus monkey(e.g., Macaca fascicularis) EGFRvIII. Other anti-EGFRvIII antibodies ofthe invention bind human EGFRvIII but do not bind, or bind only weakly,to cynomolgus monkey EGFRvIII.

Multispecific Antibodies

The antibodies of the present invention may be monospecific ormultispecific (e.g., bispecific). Multispecific antibodies may bespecific for different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244. The anti-EGFRvIII antibodies of thepresent invention can be linked to or co-expressed with anotherfunctional molecule, e.g., another peptide or protein. For example, anantibody or fragment thereof can be functionally linked (e.g., bychemical coupling, genetic fusion, noncovalent association or otherwise)to one or more other molecular entities, such as another antibody orantibody fragment to produce a bi-specific or a multispecific antibodywith a second binding specificity.

The present invention includes bispecific antibodies wherein one arm ofan immunoglobulin binds human EGFRvIII, and the other arm of theimmunoglobulin is specific for a second antigen. The EGFRvIII-bindingarm can comprise any of the HCVR/LCVR or CDR amino acid sequences as setforth in Table 1 herein. In certain embodiments, the EGFRvIII-bindingarm binds human EGFRvIII and blocks ligand binding to EGFRvIII. In otherembodiments, the EGFRvIII-binding arm binds human EGFRvIII but does notblock ligand binding to EGFRvIII.

An exemplary bispecific antibody format that can be used in the contextof the present invention involves the use of a first immunoglobulin (Ig)C_(H)3 domain and a second Ig C_(H)3 domain, wherein the first andsecond Ig C_(H)3 domains differ from one another by at least one aminoacid, and wherein at least one amino acid difference reduces binding ofthe bispecific antibody to Protein A as compared to a bi-specificantibody lacking the amino acid difference. In one embodiment, the firstIg C_(H)3 domain binds Protein A and the second Ig C_(H)3 domaincontains a mutation that reduces or abolishes Protein A binding such asan H95R modification (by IMGT exon numbering; H435R by EU numbering).The second C_(H)3 may further comprise a Y96F modification (by IMGT;Y436F by EU). Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422Iby EU) in the case of IgG4 antibodies. Variations on the bispecificantibody format described above are contemplated within the scope of thepresent invention.

Other exemplary bispecific formats that can be used in the context ofthe present invention include, without limitation, e.g., scFv-based ordiabody bispecific formats, IgG-scFv fusions, dual variable domain(DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., commonlight chain with knobs-into-holes, etc.), CrossMab, CrossFab,(SEED)body, leucine zipper, Duobody, IgG1/IgG2, dual acting Fab(DAF)-IgG, and Mab² bispecific formats (see, e.g., Klein et al. 2012,mAbs 4:6, 1-11, and references cited therein, for a review of theforegoing formats). Bispecific antibodies can also be constructed usingpeptide/nucleic acid conjugation, e.g., wherein unnatural amino acidswith orthogonal chemical reactivity are used to generate site-specificantibody-oligonucleotide conjugates which then self-assemble intomultimeric complexes with defined composition, valency and geometry.(See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012]).

Therapeutic Formulation and Administration

The invention provides pharmaceutical compositions comprising theanti-EGFRvIII antibodies or antigen-binding fragments thereof of thepresent invention. The pharmaceutical compositions of the invention areformulated with suitable carriers, excipients, and other agents thatprovide improved transfer, delivery, tolerance, and the like. Amultitude of appropriate formulations can be found in the formularyknown to all pharmaceutical chemists: Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa. These formulationsinclude, for example, powders, pastes, ointments, jellies, waxes, oils,lipids, lipid (cationic or anionic) containing vesicles (such asLIPOFECTIN™, Life Technologies, Carlsbad, Calif.), DNA conjugates,anhydrous absorption pastes, oil-in-water and water-in-oil emulsions,emulsions carbowax (polyethylene glycols of various molecular weights),semi-solid gels, and semi-solid mixtures containing carbowax. See alsoPowell et al. “Compendium of excipients for parenteral formulations” PDA(1998) J Pharm Sci Technol 52:238-311.

The dose of antibody administered to a patient may vary depending uponthe age and the size of the patient, target disease, conditions, routeof administration, and the like. The preferred dose is typicallycalculated according to body weight or body surface area. In an adultpatient, it may be advantageous to intravenously administer the antibodyof the present invention normally at a single dose of about 0.01 toabout 20 mg/kg body weight, more preferably about 0.02 to about 7, about0.03 to about 5, or about 0.05 to about 3 mg/kg body weight. Dependingon the severity of the condition, the frequency and the duration of thetreatment can be adjusted. Effective dosages and schedules foradministering anti-EGFRvIII antibodies may be determined empirically;for example, patient progress can be monitored by periodic assessment,and the dose adjusted accordingly. Moreover, interspecies scaling ofdosages can be performed using well-known methods in the art (e.g.,Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present invention include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L. P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park Ill.), to name only a few.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Fla. In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antibodies

The present invention includes methods comprising administering to asubject in need thereof a therapeutic composition comprising ananti-EGFRvIII antibody or an antibody-drug conjugate comprising ananti-EGFRvIII antibody (e.g., an anti-EGFRvIII antibody or ADCcomprising any of the HCVR/LCVR or CDR sequences as set forth in Table 1herein). The therapeutic composition can comprise any of theanti-EGFRvIII antibodies, antigen-binding fragments thereof, or ADCsdisclosed herein, and a pharmaceutically acceptable carrier or diluent.

The antibodies and ADCs of the invention are useful, inter alia, for thetreatment, prevention and/or amelioration of any disease or disorderassociated with or mediated by EGFRvIII expression or activity, ortreatable by blocking the interaction between EGFRvIII and an EGFRligand or otherwise inhibiting EGFRvIII activity and/or signaling,and/or promoting receptor internalization and/or decreasing cell surfacereceptor number. For example, the antibodies and ADCs of the presentinvention are useful for the treatment of tumors that express EGFRvIIIand/or that respond to ligand-mediated signaling. The antibodies andantigen-binding fragments of the present invention may also be used totreat primary and/or metastatic tumors arising in the brain andmeninges, oropharynx, lung and bronchial tree, gastrointestinal tract,male and female reproductive tract, muscle, bone, skin and appendages,connective tissue, spleen, immune system, blood forming cells and bonemarrow, liver and urinary tract, and special sensory organs such as theeye. In certain embodiments, the antibodies and ADCs of the inventionare used to treat one or more of the following cancers: renal cellcarcinoma, pancreatic carcinoma, head and neck cancer, prostate cancer,malignant gliomas, osteosarcoma, colorectal cancer, gastric cancer(e.g., gastric cancer with MET amplification), malignant mesothelioma,multiple myeloma, ovarian cancer, small cell lung cancer, non-small celllung cancer, synovial sarcoma, thyroid cancer, breast cancer, ormelanoma.

In the context of the methods of treatment described herein, theanti-EGFRvIII antibody may be administered as a monotherapy (i.e., asthe only therapeutic agent) or in combination with one or moreadditional therapeutic agents (examples of which are described elsewhereherein).

According to specific embodiments, the present invention providesmethods for treating a cancer, reducing tumor growth and/or causingtumor regression in a patient. The methods according to this aspect ofthe invention comprise administering to a patient a first antibody-drugconjugate (ADC) either alone or in combination with a secondanti-EGFRvIII antibody or ADC. The first ADC will typically comprise anantibody or antigen-binding fragment of an antibody and a cytotoxin,wherein the antibody or antigen-binding fragment of the first ADCspecifically binds EGFRvIII but does not bind the junctional EGFRvIIIpeptide of SEQ ID NO:148 or the peptide of SEQ ID NO:165 (i.e., thefirst ADC comprises a conformational EGFRvIII-binding antibody). Inembodiments in which a second antibody or ADC is administered, thesecond antibody or ADC will typically comprise an antibody orantigen-binding fragment of an antibody and a cytotoxin, wherein thesecond antibody or antigen-binding fragment specifically binds EGFRvIIIand also binds the junctional EGFRvIII peptide of SEQ ID NO:148 and/orthe peptide of SEQ ID NO:165 (i.e., the second antibody or ADC comprisesan EGFRvIII junctional peptide-binding antibody). When two separateanti-EGFRvIII ADCs are used in the context of this aspect of theinvention, both ADCs may, in certain embodiments, comprise the samecytotoxic agent or same class of cytotoxic agent. In other embodimentswhere two separate anti-EGFRvIII ADCs are used, each ADC may comprise adifferent cytotoxic agent and/or a different class of cytotoxic agent.Non-limiting exemplary embodiments of this aspect of the invention areset forth herein at Example 14. According to certain embodiments, theantibody or antigen-binding fragment of the first ADC (i.e., theconformational EGFRvIII binding antibody) comprises heavy and lightchain complementarity determining regions comprising SEQ ID NOs: 36, 38,40, 44, 46, and 48, or the heavy chain variable region comprising SEQ IDNO: 34 and a light chain variable region comprising SEQ ID NO:42.

Combination Therapies and Formulations

The present invention includes compositions and therapeutic formulationscomprising any of the anti-EGFRvIII antibodies described herein incombination with one or more additional therapeutically activecomponents, and methods of treatment comprising administering suchcombinations to subjects in need thereof.

The anti-EGFRvIII antibodies of the present invention may beco-formulated with and/or administered in combination with one or moreadditional therapeutically active component(s) selected from the groupconsisting of: a PRLR antagonist (e.g., an anti-PRLR antibody or smallmolecule inhibitor of PRLR), an EGFR antagonist (e.g., an anti-EGFRantibody [e.g., cetuximab or panitumumab] or small molecule inhibitor ofEGFR [e.g., gefitinib or erlotinib]), an antagonist of another EGFRfamily member such as Her2/ErbB2, ErbB3 or ErbB4 (e.g., anti-ErbB2[e.g., trastuzumab or T-DM1 {KADCYLA®}], anti-ErbB3 or anti-ErbB4antibody or small molecule inhibitor of ErbB2, ErbB3 or ErbB4 activity),a cMET antagonist (e.g., an anti-cMET antibody), an IGF1R antagonist(e.g., an anti-IGF1R antibody), a B-raf inhibitor (e.g., vemurafenib,sorafenib, GDC-0879, PLX-4720), a PDGFR-α inhibitor (e.g., ananti-PDGFR-α antibody), a PDGFR-β inhibitor (e.g., an anti-PDGFR-βantibody or small molecule kinase inhibitor such as, e.g., imatinibmesylate or sunitinib malate), a PDGF ligand inhibitor (e.g.,anti-PDGF-A, -B, -C, or -D antibody, aptamer, siRNA, etc.), a VEGFantagonist (e.g., a VEGF-Trap such as aflibercept, see, e.g., U.S. Pat.No. 7,087,411 (also referred to herein as a “VEGF-inhibiting fusionprotein”), anti-VEGF antibody (e.g., bevacizumab), a small moleculekinase inhibitor of VEGF receptor (e.g., sunitinib, sorafenib orpazopanib)), a DLL4 antagonist (e.g., an anti-DLL4 antibody disclosed inUS 2009/0142354 such as REGN421), an Ang2 antagonist (e.g., an anti-Ang2antibody disclosed in US 2011/0027286 such as H1H685P), a FOLH1antagonist (e.g., an anti-FOLH1 antibody), a STEAP1 or STEAP2 antagonist(e.g., an anti-STEAP1 antibody or an anti-STEAP2 antibody), a TMPRSS2antagonist (e.g., an anti-TMPRSS2 antibody), a MSLN antagonist (e.g., ananti-MSLN antibody), a CA9 antagonist (e.g., an anti-CA9 antibody), auroplakin antagonist (e.g., an anti-uroplakin [e.g., anti-UPK3A]antibody), a MUC16 antagonist (e.g., an anti-MUC16 antibody), a Tnantigen antagonist (e.g., an anti-Tn antibody), a CLEC12A antagonist(e.g., an anti-CLEC12A antibody), a TNFRSF17 antagonist (e.g., ananti-TNFRSF17 antibody), a LGR5 antagonist (e.g., an anti-LGR5antibody), a monovalent CD20 antagonist (e.g., a monovalent anti-CD20antibody such as rituximab), a PD-1 antibody, a PD-L1 antibody, a CD3antibody, a CTLA-4 antibody etc. Other agents that may be beneficiallyadministered in combination with the bispecific antigen-bindingmolecules of the invention include, e.g., tamoxifen, aromataseinhibitors, and cytokine inhibitors, including small-molecule cytokineinhibitors and antibodies that bind to cytokines such as IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18,or to their respective receptors.

The present invention includes compositions and therapeutic formulationscomprising any of the anti-EGFRvIII antibodies described herein incombination with one or more chemotherapeutic agents. Examples ofchemotherapeutic agents include alkylating agents such as thiotepa andcyclosphosphamide (Cytoxan™); alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziridines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine;bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone;mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinicacid; 2-ethylhydrazide; procarbazine; PSK™; razoxane; sizofiran;spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, e.g.paclitaxel (Taxol™, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddocetaxel (Taxotere™; Aventis Antony, France); chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumanalogs such as cisplatin and carboplatin; vinblastine; platinum;etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Also included in this definition areanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY 117018, onapristone, and toremifene(Fareston); and anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptablesalts, acids or derivatives of any of the above.

The anti-EGFRvIII antibodies of the invention may also be administeredand/or co-formulated in combination with antivirals, antibiotics,analgesics, corticosteroids, steroids, oxygen, antioxidants, COXinhibitors, cardioprotectants, metal chelators, IFN-gamma, and/orNSAIDs.

The additional therapeutically active component(s), e.g., any of theagents listed above or derivatives thereof, may be administered justprior to, concurrent with, or shortly after the administration of ananti-EGFRvIII antibody of the present invention; (for purposes of thepresent disclosure, such administration regimens are considered theadministration of an anti-EGFRvIII antibody “in combination with” anadditional therapeutically active component). The present inventionincludes pharmaceutical compositions in which an anti-EGFRvIII antibodyof the present invention is co-formulated with one or more of theadditional therapeutically active component(s) as described elsewhereherein.

Administration Regimens

According to certain embodiments of the present invention, multipledoses of an anti-EGFRvIII antibody (or a pharmaceutical compositioncomprising a combination of an anti-EGFRvIII antibody and any of theadditional therapeutically active agents mentioned herein) may beadministered to a subject over a defined time course. The methodsaccording to this aspect of the invention comprise sequentiallyadministering to a subject multiple doses of an anti-EGFRvIII antibodyof the invention. As used herein, “sequentially administering” meansthat each dose of anti-EGFRvIII antibody is administered to the subjectat a different point in time, e.g., on different days separated by apredetermined interval (e.g., hours, days, weeks or months). The presentinvention includes methods which comprise sequentially administering tothe patient a single initial dose of an anti-EGFRvIII antibody, followedby one or more secondary doses of the anti-EGFRvIII antibody, andoptionally followed by one or more tertiary doses of the anti-EGFRvIIIantibody.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the anti-EGFRvIII antibodyof the invention. Thus, the “initial dose” is the dose which isadministered at the beginning of the treatment regimen (also referred toas the “baseline dose”); the “secondary doses” are the doses which areadministered after the initial dose; and the “tertiary doses” are thedoses which are administered after the secondary doses. The initial,secondary, and tertiary doses may all contain the same amount ofanti-EGFRvIII antibody, but generally may differ from one another interms of frequency of administration. In certain embodiments, however,the amount of anti-EGFRvIII antibody contained in the initial, secondaryand/or tertiary doses varies from one another (e.g., adjusted up or downas appropriate) during the course of treatment. In certain embodiments,two or more (e.g., 2, 3, 4, or 5) doses are administered at thebeginning of the treatment regimen as “loading doses” followed bysubsequent doses that are administered on a less frequent basis (e.g.,“maintenance doses”).

In certain exemplary embodiments of the present invention, eachsecondary and/or tertiary dose is administered 1 to 26 (e.g., 1, 1½, 2,2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½,12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½,20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more)weeks after the immediately preceding dose. The phrase “the immediatelypreceding dose,” as used herein, means, in a sequence of multipleadministrations, the dose of anti-EGFRvIII antibody which isadministered to a patient prior to the administration of the very nextdose in the sequence with no intervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an anti-EGFRvIII antibody. For example, in certain embodiments, onlya single secondary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondarydoses are administered to the patient. Likewise, in certain embodiments,only a single tertiary dose is administered to the patient. In otherembodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiarydoses are administered to the patient. The administration regimen may becarried out indefinitely over the lifetime of a particular subject, oruntil such treatment is no longer therapeutically needed oradvantageous.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to2 weeks or 1 to 2 months after the immediately preceding dose.Similarly, in embodiments involving multiple tertiary doses, eachtertiary dose may be administered at the same frequency as the othertertiary doses. For example, each tertiary dose may be administered tothe patient 2 to 12 weeks after the immediately preceding dose. Incertain embodiments of the invention, the frequency at which thesecondary and/or tertiary doses are administered to a patient can varyover the course of the treatment regimen. The frequency ofadministration may also be adjusted during the course of treatment by aphysician depending on the needs of the individual patient followingclinical examination.

The present invention includes administration regimens in which 2 to 6loading doses are administered to a patient at a first frequency (e.g.,once a week, once every two weeks, once every three weeks, once a month,once every two months, etc.), followed by administration of two or moremaintenance doses to the patient on a less frequent basis. For example,according to this aspect of the invention, if the loading doses areadministered at a frequency of once a month, then the maintenance dosesmay be administered to the patient once every six weeks, once every twomonths, once every three months, etc.

Diagnostic Uses of the Antibodies

The anti-EGFRvIII antibodies of the present invention may also be usedto detect and/or measure EGFRvIII, or EGFRvIII-expressing cells in asample, e.g., for diagnostic purposes. For example, an anti-EGFRvIIIantibody, or fragment thereof, may be used to diagnose a condition ordisease characterized by aberrant expression (e.g., over-expression,under-expression, lack of expression, etc.) of EGFRvIII. Exemplarydiagnostic assays for EGFRvIII may comprise, e.g., contacting a sample,obtained from a patient, with an anti-EGFRvIII antibody of theinvention, wherein the anti-EGFRvIII antibody is labeled with adetectable label or reporter molecule. Alternatively, an unlabeledanti-EGFRvIII antibody can be used in diagnostic applications incombination with a secondary antibody which is itself detectablylabeled. The detectable label or reporter molecule can be aradioisotope, such as ³H, ¹⁴C, ³²S, or ¹²⁵I; a fluorescent orchemiluminescent moiety such as fluorescein isothiocyanate, orrhodamine; or an enzyme such as alkaline phosphatase,beta-galactosidase, horseradish peroxidase, or luciferase. Specificexemplary assays that can be used to detect or measure EGFRvIII in asample include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in EGFRvIII diagnostic assays according to thepresent invention include any tissue or fluid sample obtainable from apatient which contains detectable quantities of EGFRvIII protein, orfragments thereof, under normal or pathological conditions. Generally,levels of EGFRvIII in a particular sample obtained from a healthypatient (e.g., a patient not afflicted with a disease or conditionassociated with abnormal EGFRvIII levels or activity) will be measuredto initially establish a baseline, or standard, level of EGFRvIII. Thisbaseline level of EGFRvIII can then be compared against the levels ofEGFRvIII measured in samples obtained from individuals suspected ofhaving a EGFRvIII related disease or condition.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1. Generation of Anti-EGFRvIII Antibodies

Anti-EGFRvIII antibodies were obtained by immunizing a VELOCIMMUNE®mouse (i.e., an engineered mouse comprising DNA encoding humanimmunoglobulin heavy and kappa light chain variable regions) with animmunogen comprising the extracellular domain of EGFRvIII. Antibodies ofthe first set include the antibodies designated as H1H2194P, H1H2195P,H2M1863N2, H2M1911N, H2M1912N, H2M1915N, H2M1917N, H2M1918N, andH3M1913N (as shown in Tables 1 and 2).

The antibody immune response was monitored by an EGFRvIII-specificimmunoassay. When a desired immune response was achieved splenocyteswere harvested and fused with mouse myeloma cells to preserve theirviability and form hybridoma cell lines. The hybridoma cell lines werescreened and selected to identify cell lines that produceEGFRvIII-specific antibodies. Using this technique several anti-EGFRvIIIchimeric antibodies (i.e., antibodies possessing human variable domainsand mouse constant domains) were obtained. In addition, several fullyhuman anti-EGFRvIII antibodies were isolated directly fromantigen-positive B cells without fusion to myeloma cells, as describedin US 2007/0280945A1.

Separately, H1H1863N2 with reduced fucosylation [“H1H1863N2(Fuc−)”] wasalso prepared in a CHO host cell line that was described as “8088” in USPatent Application No. 2010/0304436A1, which is specificallyincorporated by reference in its entirety. Briefly, the light chain andheavy chain sequences of H1H1863N2 were cloned into expression vectors.Two million 8088 cells were transfected with the light and heavy chainplasmids, and pR4004 vector containing the gene encoding Cre.Transfected cells that survived selection with 400 μg/ml hygromycin wereadapted to grow in suspension in serum-free, fucose-free medium. Cellsthat expressed fluorescent protein EGFP but not DsRed or ECFP from thetransfected cells were isolated by flow cytometry. The sorted cells wereseeded in a shaker flask at 4×10⁵ cells/ml and, three days later, theculture medium was collected and the antibody protein therein [i.e.,H1H1863N2(Fuc−)] was purified by Protein A chromatography. Massspectrometry analysis of the resulting H1H1863N2(Fuc−) confirmed thatcore fucose was removed relative to the H1H1863N2(Fuc+), originalantibody. The designations, “H1H1863N2” and “H1H1863N2(Fuc+)” herein,both indicate the original antibody without fucosylation modifications.

Certain biological properties of the exemplary anti-EGFRvIII antibodiesgenerated in accordance with the methods of this Example are describedin detail in the Examples set forth below.

Example 2. Heavy and Light Chain Variable Region Amino Acid and NucleicAcid Sequences

Table 1 sets forth the amino acid sequence identifiers of the heavy andlight chain variable regions and CDRs of selected anti-EGFRvIIIantibodies of the invention. The corresponding nucleic acid sequenceidentifiers are set forth in Table 2.

TABLE 1 Amino Acid Sequence Identifiers Antibody SEQ ID NOs: DesignationHCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H2194P 2 4 6 8 10 12 1416 H1H2195P 18 20 22 24 26 28 30 32 H2M1863N2 34 36 38 40 42 44 46 48H2M1911N 50 52 54 56 58 60 62 64 H2M1912N 66 68 70 72 74 76 78 80H2M1915N 82 84 86 88 90 92 94 96 H2M1917N 98 100 102 104 106 108 110 112H2M1918N 114 116 118 120 122 124 126 128 H3M1913N 130 132 134 136 138140 142 144

TABLE 2 Nucleic Acid Sequence Identifiers Antibody SEQ ID NOs:Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 H1H2194P 1 3 57 9 11 13 15 H1H2195P 17 19 21 23 25 27 29 31 H2M1863N2 33 35 37 39 4143 45 47 H2M1911N 49 51 53 55 57 59 61 63 H2M1912N 65 67 69 71 73 75 7779 H2M1915N 81 83 85 87 89 91 93 95 H2M1917N 97 99 101 103 105 107 109111 H2M1918N 113 115 117 119 121 123 125 127 H3M1913N 129 131 133 135137 139 141 143

Antibodies are typically referred to herein according to the followingnomenclature: Fc prefix (e.g. “H1H,” “H2M,” “H3M,” etc.), followed by anumerical identifier (e.g. “2194,” “2195,” “1863,” etc.), followed by a“P” or “N” suffix, as shown in Tables 1 and 2. Thus, according to thisnomenclature, an antibody may be referred to herein as, e.g.,“H1H2194N,” “H2M1911N,” “H3M1913N,” etc. The H1H, H2M and H3M prefixeson the antibody designations used herein indicate the particular Fcregion isotype of the antibody. For example, an “H1H” antibody has ahuman IgG1 Fc, an “H2M” antibody has a mouse IgG2 Fc, and an “H3M”antibody has a mouse IgG3 Fc, (all variable regions are fully human asdenoted by the first ‘H’ in the antibody designation). As will beappreciated by a person of ordinary skill in the art, an antibody havinga particular Fc isotype can be converted to an antibody with a differentFc isotype (e.g., an antibody with a mouse IgG1 Fc can be converted toan antibody with a human IgG4, etc.), but in any event, the variabledomains (including the CDRs)—which are indicated by the numericalidentifiers shown in Tables 1 and 2—will remain the same, and thebinding properties are expected to be identical or substantially similarregardless of the nature of the Fc domain.

Control Constructs Used in the Following Examples

Control constructs were included in the following experiments forcomparative purposes: Control I: Human anti-EGFRvIII antibody (IgG1)with heavy and light chain variable domains having the amino acidsequences corresponding to SEQ ID NOS:142 and 144, respectively, of the“13.1.2” antibody disclosed in U.S. Pat. No. 7,736,644; Control II:Chimeric anti-EGFRvIII antibody (hIgG1) with heavy and light chainvariable domains having the amino acid sequences corresponding to SEQ IDNOS:11 and 12, respectively, of the “ch806” antibody disclosed in U.S.Pat. No. 7,589,180; Control III: Humanized anti-EGFRvIII antibody(hIgG1) with heavy and light chain variable domains having the aminoacid sequences corresponding to SEQ ID NOS:42 and 47, respectively, ofthe “hu806” antibody disclosed in US Patent Application Publication No.2010/0056762; Control IV: a chimeric anti-EGFR antibody with heavy andlight chain variable domains having the amino acid sequences of thecorresponding domains of “C225,” as set forth in U.S. Pat. No.7,060,808; and Control V: Human anti-EGFRvIII antibody (IgG1) with heavyand light chain variable domains having the amino acid sequencescorresponding to SEQ ID NOS: 2 and 19, respectively, of the “131”antibody of U.S. Pat. No. 7,736,644 B2. The “13.1.2” antibody is knownto be specific for the junctional peptide (SEQ ID NO:148) of EGFRvIII;and the “ch806” and “hu806” antibodies are known to bind to residues311-326 (SEQ ID NO:165) of EGFR (SEQ ID NO:146), which is amplified oroverexpressed, or residues 44-59 of EGFRvIII (SEQ ID NO:147).

Example 3. EGFRvIII Binding Affinity Determination

Binding affinities and kinetic constants of human monoclonalanti-EGFRvIII antibodies were determined by surface plasmon resonance at37° C. Measurements were conducted on a T100 BIACORE™ instrument.Antibodies, expressed as human IgG1 Fc (i.e., “H1H” designations), werecaptured onto an anti-human Fc sensor surface (mAb-capture format), andsoluble monomeric [EGFR-mmh (SEQ ID NO:154) and EGFRvIII-mmh (SEQ IDNO:152)] or dimeric [EGFR-mFc (SEQ ID NO:155) and EGFRvIII-mFc (SEQ IDNO:153)] proteins were injected over the surface. In thereceptor-capture format, either EGFRvIII-mFc or EGFR-mFc, was capturedon the BIACORE™ chip and the respective antibodies flowed over. Kineticassociation (k_(a)) and dissociation (k_(d)) rate constants weredetermined by processing and fitting the data to a 1:1 binding modelusing Scrubber 2.0 curve fitting software. Binding dissociationequilibrium constants (K_(D)) and dissociative half-lives (t_(1/2)) werecalculated from the kinetic rate constants as: K_(D) (M)=k_(d)/k_(a);and t₁₁₂ (min)=ln2/(60*k_(d)).

Results are shown in Tables 3 and 4. NB=no binding under the conditionstested; NT=not tested.

TABLE 3 (Binding kinetics of human Fc antibodies) Binding at 37°C./MAb-Capture Format Ab Analyte k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) T½H1H1863N2 EGFRvIII-mmh 1.97E+04 8.95E−03 4.54E−07 1.3 (Fuc+) EGFR-mmh NTNT NT NT EGFRvIII-mFc 7.28E+04 8.07E−04 1.11E−08 14 EGFR-mFc NT NT NT NTH1H1863N2 EGFRvIII-mmh 3.02E+04 1.02E−02 3.39E−07 1.1 (Fuc−) EGFR-mmh NBNB NB NB EGFRvIII-mFc 1.12E+05 6.42E−04 5.73E−09 18 EGFR-mFc NB NB NB NBH1H1911N EGFRvIII-mmh NB NB NB NB EGFR-mmh NB NB NB NB EGFRvIII-mFc NBNB NB NB EGFR-mFc NB NB NB NB H1H1912N EGFRvIII-mmh 1.83E+04 1.64E−028.99E−07 0.7 EGFR-mmh NB NB NB NB EGFRvIII-mFc 2.04E+04 9.71E−044.77E−08 12 EGFR-mFc NB NB NB NB H1H1913N EGFRvIII-mmh 1.63E+02 1.14E−037.03E−06 10 EGFR-mmh NB NB NB NB EGFRvIII-mFc 1.40E+04 3.16E−04 2.26E−0837 EGFR-mFc NB NB NB NB H1H1915N EGFRvIII-mmh NB NB NB NB EGFR-mmh NB NBNB NB EGFRvIII-mFc NB NB NB NB EGFR-mFc NB NB NB NB H1H2194PEGFRvIII-mmh 8.10E+04 1.37E−03 1.70E−08 8 EGFR-mmh 7.60E+04 9.60E−041.26E−08 12 EGFRvIII-mFc 9.54E+04 2.22E−04 2.33E−09 52 EGFR-mFc 8.10E+041.99E−04 2.43E−09 58 H1H2195P EGFRvIII-mmh 6.48E+04 6.94E−04 1.07E−08 17EGFR-mmh 5.66E+04 5.23E−04 9.20E−09 22 EGFRvIII-mFc 1.02E+05 1.13E−041.10E−09 103 EGFR-mFc 9.20E+04 1.89E−04 2.05E−09 61 Control IEGFRvIII-mmh 1.29E+05 1.53E−01 1.19E−06 0.1 EGFR-mmh NB NB NB NBEGFRvIII-mFc 7.15E+04 7.36E−03 1.03E−07 1.6 EGFR-mFc NB NB NB NB ControlII EGFRvIII-mmh 4.90E+04 7.33E−03 1.50E−07 2 EGFR-mmh NB NB NB NBEGFRvIII-mFc 2.02E+05 4.08E−04 2.02E−09 28 EGFR-mFc NB NB NB NB ControlIII EGFRvIII-mmh 8.57E+04 5.16E−03 6.02E−08 2.2 EGFR-mmh NB NB NB NBEGFRvIII-mFc 2.52E+05 2.98E−04 1.18E−09 39 EGFR-mFc NB NB NB NB ControlV EGFRvIII-mmh 1.94E+05 1.59E−02 8.20E−08 1 EGFR-mmh NB NB NB NBEGFRvIII-mFc 1.91E+05 3.71E−04 1.95E−09 31 EGFR-mFc NT NT NT NT

TABLE 4 (Binding kinetics of human Fc antibodies) Binding at 37°C./Receptor-Capture Format Receptor ka kd K_(D) Ab Captured (M⁻¹s⁻¹)(s⁻¹) (M) T½ H1H1863N2 EGFRvIII- 9.00E+05 2.06E−04 2.30E−10 56 (Fuc+)mFc EGFR-mFc 2.11E+05 1.82E−01 8.65E−07 0.1 H1H1863N2 EGFRvIII- 1.01E+062.15E−04 2.10E−10 54 (Fuc−) mFc EGFR-mFc 1.99E+05 4.67E−01 2.34E−06 0.02H1H1911N EGFRvIII- 3.29E+04 6.43E−04 1.95E−08 18 mFc EGFR-mFc 7.77E+031.74E−03 2.24E−07 7 H1H1912N EGFRvIII- 9.90E+04 5.37E−04 5.40E−09 22 mFcEGFR-mFc 3.99E+04 9.14E−04 2.29E−08 13 H1H1913N EGFRvIII- 6.30E+041.00E−06 1.58E−11 11550 mFc EGFR-mFc 5.93E+03 1.00E−06 1.69E−10 11550H1H1915N EGFRvIII- 1.00E+05 3.28E−04 3.20E−09 35 mFc EGFR-mFc 4.35E+048.01E−03 1.84E−07 1.4 H1H2193N EGFRvIII- 2.17E+05 5.85E−05 2.68E−10 197mFc EGFR-mFc 2.04E+05 9.15E−05 4.47E−10 126 H1H2194N EGFRvIII- 1.88E+057.38E−05 3.94E−10 157 mFc EGFR-mFc 1.87E+05 7.07E−05 3.80E−10 163H1H2195N EGFRvIII- 2.37E+05 2.53E−05 1.06E−10 456 mFc EGFR-mFc 2.25E+055.20E−05 2.31E−10 222 Control I EGFRvIII- 4.46E+05 4.04E−03 9.06E−09 2.9mFc EGFR-mFc NB NB NB NB Control II EGFRvIII- 1.25E+06 7.31E−05 5.90E−11158 mFc EGFR-mFc 4.44E+05 1.46E−04 3.29E−10 79 Control III EGFRvIII-1.49E+06 1.00E−06 6.70E−13 11550 mFc EGFR-mFc 2.86E+05 6.17E−05 2.15E−10187

As shown in Tables 3 and 4, several antibodies showed selectivity forEGFRvIII and did not bind wild-type EGFR in the mAb-capture format. Inthe receptor capture format (Table 4) H1H863N2, H1H1915N and Control Ishowed the greatest selectivity.

Experiment 4: Antibody Specificity Determined by ELISA

To further characterize anti-hEGFRvIII mAbs, their binding specificitywas examined by ELISA. Plates were coated with one of the following:EGFR-mmh (SEQ ID NO:154); EGFRvIII-mmh (SEQ ID NO:152); and a junctionalpeptide (J-peptide) (SEQ ID NO:148). For the junctional peptides thatwere linked to biotin either at C-terminal (SEQ ID NO:149) or N-terminal(SEQ ID NO:150) via a linker, plates were pre-coated with avidin. Also,coated was an irrelevant peptide (control peptide) with or withoutbiotin at its N-terminal. Anti-EGFRvIII antibodies as well as an isotypecontrol antibody were added to coated plates and allowed to incubate for1 hour at 25° C. The plates were then washed and bound anti-EGFRvIIImAbs were detected with anti-human Fc antibodies conjugated withhorse-radish peroxidase (HRP). Plates were developed with atetra-methyl-benzidine (TMB) substrate solution to produce acolorimetric reaction and neutralized with sulfuric acid before readingabsorbance at 450 nm on a VICTOR™ X5 plate reader. Data analysis used asigmoidal dose-response model within PRISM™ software. The calculatedEC₅₀ value, defined as 50% of antibody concentration required to developmaximal response, was used as an indicator of binding potency. Theresults are shown in Table 5. NT: Not tested. Controls I-III: Asdescribed above.

TABLE 5 EC50 (nM) N-term EGFR- EGFRvIII- Biotin mmh mmh C-term biotinN-term biotin Control control Antibody (25° C.) (25° C.) J-peptideJ-peptide J-peptide peptide peptide H1H1863N >100.0766 >10 >10 >10 >10 >10 2 (Fuc−) H1H1863N >100.113 >10 >10 >10 >10 >10 2 (Fuc+) H1H1911N 9.060.0748 >10 >10 >10 >10 >10 H1H1912N 0.0405 0.0118 >10 >10 >10 >10 >10H1H1913N 2.55 2.14 >10 >10 >10 >10 >10 H1H1915N >100.167 >10 >10 >10 >10 >10 H1H2193P 0.0040 0.0035 >10 >10 >10 >10 >10H1H2194P 0.0037 0.0032 >10 >10 >10 >10 >10 H1H2195P 0.00520.0049 >10 >10 >10 >10 >10 Control I >10 0.0094 0.118 0.01530.0106 >10 >10 Control II 0.0095 0.0057 >10 >10 >10 >10 >10 Control III0.0079 0.0048 NT NT NT NT NT Isotype >10 >10 >10 >10 >10 >10 >10 Control

Antibodies H1H1863N2, H1H1915 and Control I showed strong binding toEGFRvIII but no binding (>10 nM) to wild-type EGFR. None of theantibodies, except Control I (having the sequences that correspond tothe heavy and light chain sequences of the “13.1.2” antibody derivedfrom mice immunized with junctional peptide (U.S. Pat. No. 7,736,644),showed binding to the junctional peptides.

Example 5. Western Blot of EGFR and EGFRvIII Using Anti-EGFRvIIIAntibodies

One of the antibodies, H1H1863N2, was tested for its bindingcharacteristics with western blots under both reduced and non-reducedconditions. EGFR-mmh (SEQ ID NO:154) or EGFRvIII-mmh (SEQ ID NO:152) wasloaded onto Tris-Glycine SDS PAGE gels, run and then transferred tonitrocellulose. After blocking, membranes were cut in half and probedwith either anti-EGFRvIII antibodies or anti-His antibody. Controls Iand II are as described above.

As shown in FIG. 1a , H1H1862N2 (Fuc−) does not bind reduced ornon-reduced EGFRvIII-mmh or EGFR-mmh and thus has a conformationalepitope to EGFRvIII. In contrast, Control II binds both wildtype andvariant III EGFR under reduced and non-reduced conditions, while ControlI, a junctional peptide binder, is specific for EGFRvIII. Both Control Iand II, in contrast to H1H1863N2, have linear binding epitopes. FIG. 1bshows other EGFRvIII antibodies, which show mixed behaviors on Westernblots.

Example 6. EGFR/EGFRvIII Peptide Binding and Antibody Competition Assays

H1H1863N2(Fuc−) was tested for its binding characteristics using peptidebinding and antibody competition assays. For peptide binding experimentsthe EGFRvIII junctional peptide (SEQ ID NO:148) tagged via a linker withbiotin at its C-terminus [i.e., LEEKKGNYVVTDHGGGGSK (SEQ IDNO:149)-biotin] or the peptide consisting of residues 311-326 of EGFR(the “EGFR 311-326 peptide”; SEQ ID NO:165) tagged via a linker withbiotin at its C-terminus [i.e., CGADSYEMEEDGVRKCGGGGSK (SEQ IDNO:151)-biotin] were captured to ˜0.4 nM of thickness using streptavidincoated OCTET® tips on a FORTEBIO® OCTET® RED instrument. After peptidecapture, the coated tips were placed in 1 μM solutions of antibody andthe binding responses were recorded (see FIG. 2). Controls I-III are thesame as those described above.

As predicted Control I bound the junctional peptide with C-terminalbiotin and Controls II and III bound the EGFR 311-326 peptide withC-terminal biotin. H1H1863N2(Fuc−) failed to bind either of thepeptides.

For antibody cross competition, ˜200 resonance units (RU) ofhEGFRvIII-mmh (SEQ ID NO:152) was captured onto a BIACORE™ surfacecoated with a high-density, anti-penta-Histidine polyclonal antibody(cat. #34660, QUIAGEN). Using a coinjection methodology, capturedhEGFRvIII-mmh was saturated by a 5-minute injection of 500 nM of a firstmAb immediately followed by another 5-minute injection of a second mAb(500 nM) which was supplemented with 500 nM of the first mAb.Significant binding, expressed as RU, of the second mAb was interpretedthat it does not compete for binding with the first mAb. For controlexperiments isotype matched mAbs were used as either a first mAb or asecond mAb. Results are shown in Table 6.

TABLE 6 Second Antibody Binding (RU) BIACORE ™ H1H1863N2 Control IControl II Control III Surface (Fuc−) Binding Binding Binding Binding(First Antibody) Response Response Response Response EGFRvIII alone 270234 247 247 EGFRvIII - 5 253 191 208 H1H1863N2(Fuc−) Complex EGFRvIII -291 5 258 272 Control I Complex EGFRvIII - 225 252 6 25 Control IIComplex EGFRvIII - 223 254 13 7 Control III Complex

H1H1863N2(Fuc−) did not compete with any of control antibodies I-III forbinding to the hEGFRvIII-mmh capture surface. As expected controls IIand III, both of which are known to bind to residues 311-326 of EGFR,competed with each other for binding to the EGFRvIII-mmh capturesurface.

Example 7. Cell Binding Selectivity of Anti-EGFRvIII Antibodies

To determine the specificity of the anti-EGFRvIII mAbs, their binding toHEK293, HEK293 cells expressing EGFRvIII (HEK293/EGFRvIII) and A431cells, was analyzed by fluorescence activated cell sorting (FACS).HEK293/EGFRvIII cells were prepared by transfecting HEK293 cells withneomycin resistant DNA vectors constitutively expressing full-lengthhEGFRvIII (SEQ ID NO:147) using LIPOFECTAMINE™ 2000 transfection reagent(INVITROGEN™). At two days post-transfection, cells were placed underG418 selection for approximately two weeks. Populations positivelyexpressing EGFRvIII were isolated via fluorescence activated cellsorting (FACS). The HEK293 cells expressing ˜3×10⁶ copies of EGFRvIIIper cell were used in the experiment. Briefly, the anti-EGFRvIIIantibodies at 10 μg/ml were incubated with cells for 30 minutes at roomtemperature, washed, incubated with secondary antibody, i.e.,phycoerythrin (PE)-labeled goat F(ab′)₂ against human IgG (cat#109-116-170, Jackson ImmunoResearch Laboratories), followed by a finalwash before FACS analysis. In another set of experiment, anti-EGFRvIIIantibodies were directly conjugated via their lysine residues with thefluorescent dye, ALEXA FLUOR® 488 Dye (INVITROGEN™), thereby eliminatingthe step using the secondary antibody. The results from HEK293 cells andHEK293/EGFRvIII cells using directly labeled anti-EGFRvIII antibodiesare shown in Table 7 and those using the secondary PE-labeled anti-Fc(human or mouse) are shown in Table 8. The results from A431 cells usingdirectly labeled anti-EGFRvIII antibodies are shown in Table 9 and thoseusing the secondary PE-labeled anti-Fc (human or mouse) are shown inTable 10. Controls I, II, III, IV and V are described above. MFI: MeanFluorescence Intensity.

TABLE 7 Parental HEK293/ Ratio HEK293 EGFRvIII (EGFRvIII MFI/ AntibodyMFI MFI parental MFI) Unstained 3548 4005 1.1 H1H1863N2 (Fuc−) 3776361000 95.6 H1H1863N2 (Fuc+) 3805 360000 94.6 H1H1911N 3593 55064 15.3H1H1912N 3727 122000 32.7 H1H1913N 4801 239000 49.8 H1H1915N 3461 7341321.2 Control I 3559 258000 72.5 Control II 3582 313000 87.4 Control IV24954 439000 17.6

TABLE 8 Parental HEK293/ Ratio HEK293 EGFRvIII (EGFRvIII MFI/ AntibodyMFI MFI parental MFI) Unstained 819 920 1.1 PE anti-human IgG 1027 11061.1 H1H1863N2 (Fuc−) 1671 301000 180.1 H1H1911N 1812 107000 59.1H1H2194P 981 18583 18.9 H1H2195P 1176 13517 11.5 Control I 1480 272000183.8 Control II 1015 313000 308.4 Control IV 23325 354000 15.2 ControlV 11732 997062 85.0

TABLE 9 Fold Above Antibody A431 MFI Background Unstained 6708 1.0H1H1863N2 (Fuc−) 26036 3.9 H1H1911N 15984 2.4 H1H1912N 14343 2.1H1H1915N 8440 1.2 Control I 9652 1.4 Control II 15716 2.3 Control III71514 10.7 Control IV 962000 143.4

TABLE 10 Fold Above Antibody A431 MFI Background Unstained 1314 0.9 PEanti-human IgG 1428 1.0 H1H1863N2 (Fuc−) 3385 2.4 H1H1911N 3140 2.2H1H2194P 2291 1.6 H1H2195P 2227 1.6 Control I 1448 1.0 Control II 55763.9 Control IV 395000 276.6 Control V 4240 3.0

Several anti-EGFRvIII antibodies showed a distinct binding preferencefor the HEK293/EGFRVIII cell line over the parental HEK293 cells wheneither detected using directly labeled anti-EGFRvIII antibodies (Table7) or a secondary PE labeled anti-human IgG (Table 8). Most antibodieswhen incubated with A431 cells (30 minutes at 4° C.) displayed minimalto no binding, except for Controls III and IV antibodies (Tables 9 and10).

Example 8. Internalization of Anti-EGFRvIII mAbs by HEK293/EGFRvIIICells

Anti-EGFRvIII mAbs (10 ug/ml) were incubated with HEK293/EGFRVIII (seeExample 7, supra) cells for 2 hours on ice followed by two PBS washes.Cells were then subjected to a 30-min incubation on ice with secondaryDYLIGHT™ 488-conjugated anti-human IgG Fab fragments (JacksonImmunoResearch Laboratories) followed by two additional PBS washes.Antibodies were allowed to internalize for 1 h at 37° C. ininternalization buffer (PBS+FBS) or remained at 4° C. Cells were fixedin 4% formaldehyde, and nuclei stained with DRAQ5® DNA dye (CellSignaling Technology, Inc.). Images were acquired at 40× on theIMAGEXPRESS™ high content system (Molecular Devices) and internalizedvesicles were quantitated using Columbus software (Perkin Elmer). Theresults are shown in Tables 11 and FIG. 3.

TABLE 11 Fluorescent Intensity of Fluorescent Intensity of vesicles 4°C. vesicles 37° C. Ab Mean ±SD Mean ±SD H1H1863N2(Fuc−) 29896 8333617184 46823 H1H1911N 29834 11879 280439 61121 H1H1912N 4912 1774 37020112205 Control I 21981 4613 263506 28067 Control II 20339 5644 615239144397 Control IV 92311 19386 1078196 106073

Robust internalization occurred at 37° C. for H1H1863N2, Control II, andControl IV. Internalization was also observed for H1H1911N, H1H1912N andControl I.

Example 9. Binding of Anti-EGFRvIII Antibody to U87/EGFRvIII TumorXenograft

To further determine the specificity of H1H1863N2, human glioblastomacell line U87 expressing EGFRvIII was prepared as described forHEK293/EGFRvIII cells in Example 7. U87 cells expressing ˜1.5×10⁵ copiesof EGFRvIII per cell (U87/EGFRvIII) were used in the experiment.U87/EGFRvIII cells (3×10⁶ cells) were xenografted in severe combinedimmunodeficient (SCID) mice and tumors were allowed to grow until amedian size of 200-300 mm³ was obtained. Mice were then injected withH1H1863N2(Fuc−) or isotype control via tail vein. At 10 minutes, 4 hoursand 24 hours post injection of the antibody, mice were sacrificed andtumors were removed and placed into PBS. Tumors were immediatelydissociated and stained with an allophycocyanin (APC)-conjugatedanti-human Fc (hFc-APC) antibody. Stained cells were washed 3 times withflow PBS containing 2% fetal calf serum and 0.1% sodium azide. Tumors atthe 10-min and 4-hour time points were fixed overnight and then measuredby flow cytometer. Tumors collected at 24-hour time point were measuredwithout being fixed. All samples were collected on an ACCUR1® C6 FLOWCYTOMETER® (Accuri Cytometers, Inc.) and the mean fluorescence intensity(MFI) determined. The results are shown in Table 12. MFI values are theaverage of 2-3 biological replicates ±the standard error of the mean(SEM).

TABLE 12 Time MFI ± SEM (U87/EGFRvIII) Post-Injection Isotype ControlH1H1863N2(Fuc−) 10 minutes 708 ± 4  2259 ± 115 4 hours 741 ± 34 10620 ±2881 24 hours 664 ± 34 27923 ± 3297

Compared to isotype-control, H1H1863N2(Fuc−) antibody bound U87/EGFRvIIItumor cells efficiently in a time-dependent manner.

Example 10. Binding of Anti-EGFRvIII Antibody to B16F10.9/EGFRvIII TumorXenograft

SCID mice were implanted with fifty thousand of murine melanoma cellsB16F10.9 or B16F10.9 over-expressing EGFRvIII (B16F10.9/EGFRvIII).B16F10.9/EGFRvIII cells were prepared as described for HEK293/EGFRvIIIcells in Example 7. B16F10.9 cells expressing ˜1.5×10⁵ copies ofEGFRvIII per cell are used for this experiment. Tumors were allowed togrow for approximately 14 days, until a median size of 200-300 mm³ wasobtained. Mice were then injected with H1H1863N2(Fuc−) or isotypecontrol via their tail vein. At 10 minutes, 4 hours and 24 hours postinjection of antibody, mice were sacrificed and tumors were removed andplaced into PBS. Tumors were immediately dissociated and stained with anallophycocyanin conjugated anti-human Fc (hFc-APC) antibody. Stainedcells were washed 3× with flow PBS (1×PBS, 2% fetal calf serum, 0.1%sodium azide), fixed and permealized using standard methods. Flowcytometry was used to detect cell surface-bound H1H1863N2(Fuc−) andanalysis was performed using FlowJo software (Tree Star, Inc.). Theresults are shown in Table 13 and FIG. 4a . To detect both cellsurface-bound and intracellularly-bound antibodies, cells were stained asecond time using the same anti-human Fc (hFc-APC) antibody followingthe fixation and permeabilization steps. This allowed for intracellularantibody to be detected. The results are shown in Table 14 and FIG. 4b .All samples were collected on an ACCURI® C6 FLOW CYTOMETER® and the meanfluorescence intensity (MFI) determined. MFI for each sample wasreported after subtracting the MFI of the unstained control. MFI valuesare the average of two biological replicates (N=2) ±the standard errorof the mean (SEM).*N=1 for this time point.

TABLE 13 MFI ± SEM (B16F10.9/EGFRvIII) - Surface Staining B16F10.9B16F10.9/EGFRvIII Time Isotype H1H1863N2 Isotype H1H1863N2Post-Injection Control (Fuc−) Control (Fuc−) 10 minutes 74 ± 67 56 ± 2128 ± 49  2003 ± 216 4 hour 80 ± 15 195 ± 52 54 ± 21 4224 ± 610 24 hour79 ± 21 155 ± 42 72* 5692 ± 595

TABLE 14 MFI ± SEM (B16F10.9/EGFRvIII) - Surface & Internal StainingB16F10.9 B16F10.9/EGFRvIII Time Isotype H1H1863N2 Isotype H1H1863N2Post-Injection Control (Fuc−) Control (Fuc−) 10 minutes 132 ± 92 117 ±18  155 ± 44 2627 ± 192 4 hour 165 ± 22 422 ± 106 120 ± 22 7785 ± 782 24hour 135 ± 11 281 ± 51  132* 9578 ± 852

H1H1863N2(Fuc−) bound efficiently to the surface of B16F10.9 cellsexpressing EGFRvIII in a time-dependent manner, while the binding ofisotype control was minimal. The increase in total binding (i.e., cellsurface bound plus internally bound) of H1H1863N2(Fuc−), compared to itsbinding to cell surface only, indicated that the cell surface-boundantibodies were effectively internalized by B16F10.0 cells.

Example 11. Pharmacokinetics of Anti-EGFRvIII Antibodies in Mice

To determine the in vivo selectivity of anti-EGFRvIII antibodies apharmacokinetic study using wild-type mice (“WT mice”) naturallyexpressing mouse EGFR, and humanized EGFR mice (“hEGFR mice”) expressinghuman EGFR, was carried out. Mice were from cross-bred strains with abackground containing C57BL6 (75%) and 129Sv (25%). Cohorts contained 5each of either WT or hEGFR mice. All antibodies were administeredsubcutaneously at a dose of 0.2 mg/kg. Bleeds were collected at 0 hour,6 hours, 1 day, 2 days, 3 days, 4 days, 7 days, 10 days, 14 days, 21days, and 30 days after the administration. Serum levels of humanantibodies were determined by sandwich ELISA. Briefly, a goat polyclonalanti-human IgG (Fc-specific) antibody (Jackson ImmunoResearch) wascoated in 96-well plates at a concentration of one μg/ml and incubatedovernight at 4° C. After the plates were blocked with BSA, serum samplesin six-dose serial dilutions and reference standards of the respectiveantibodies in twelve-dose serial dilutions were added to the plate andincubated for one hour at room temperature. After washing to removeunbound antibody, captured human antibodies were detected using the samegoat polyclonal anti-human IgG (Fc-specific) antibody conjugated withhorseradish peroxidase (HRP) (Jackson ImmunoResearch) and developed bystandard colorimetric tetramethylbenzidine (TMB) substrate according tothe manufacturer's recommendation. Absorbances at 450 nm were recordedon a plate reader and the concentration of hIgG in serum samples werecalculated using the reference standard curve generated in the sampleplate. Mouse anti-human antibodies (MAHA) were measured using standardmethods and were generally low.

FIGS. 5a-5d show the antibody concentration vs. time plots for the fourtested antibodies. Control IV (“Mab C225”) is known to bind human EGFRbut not its mouse homologue. As expected, this antibody displayed fastclearance in hEGFR mice and slow clearance (i.e., no target-mediatedclearance) in WT mice (FIG. 5a ). Control I (“Mab 13.1.2”) is known tobind the EGFRvIII junctional peptide “LEEKKGNYVVTDH” that is not presentin human or mouse EGFR. The antibody does not bind human or mouse EGFRin vivo. As expected, this antibody displayed identical slowpharmacokinetic clearance rates in both types of mice (FIG. 5b ) and notarget-mediated clearance was observed. Control III antibody (“Mabhu806”) showed increased clearance in hEGFR mice relative to WT mice(FIG. 5c ). This finding is consistent with its ability to bind hEGFR invitro as determined by Biacore (see Example 3, Table 4) and FACS(Example 7, Table 9). FIG. 5d shows the clearance of H1H1863N2(Fuc+).This antibody, similar to control I, displayed identical slow clearancerates in both types of mice. Thus, H1H1863N2 does not bind human ormouse EGFR in vivo.

Example 12. An Anti-EGFRvIII Antibody-Drug Conjugate Inhibits TumorGrowth in In Vivo EGFRvIII-Positive Breast Cancer Allograft Models

In this Example, two different antibody-drug conjugates of the exemplaryanti-EGFRvIII antibody H1H1863N2 were tested for their ability toinhibit tumor growth in vivo. A first ADC was produced by conjugatingH1H1863N2 to the maytansinoid toxin DM1 via a non-cleavable MCC linker(see, e.g., U.S. Pat. No. 5,208,020 and US application 2010/0129314) toproduce “H1H1863N2-MCC-DM1.” A second ADC was produced by conjugatingH1H1863N2 to a modified version of DM1 attached to a novel cleavablelinker, referred to as “M0026” (also known as “compound 7” inWO2014/145090, the disclosure of which is incorporated by referenceherein in its entirety), to yield “H1H1863N2-M0026.” When tested forcytotoxicity in vitro against MMT/EGFRvIII cells, H1H1863N2-MCC-DM1exhibited an IC₅₀ of 12 nM whereas H1H1863N2-7 exhibited an IC₅₀ of 0.8nM based on drug equivalents.

To compare the in vivo efficacy of the anti-EGFRvIII antibodiesconjugated to DM1 and M0026, studies were performed in immunocompromisedmice bearing EGFRvIII positive breast cancer allografts.

Briefly, tumor allografts were established by subcutaneous implantationof 0.5×10⁶ MMT/EGFRvIII cells into the left flank of female CB17 SCIDmice (Taconic, Hudson, N.Y.). Once tumors had reached an average volumeof 140 mm³ (˜Day 8), mice were randomized into groups of seven, anddosed with anti-EGFRvIII ADCs using either the MCC-DM1 or M0026linker-drug format. Control reagents, including non-binding ADCs usingeither the MCC-DM1 or M0026 linker-drug format, and PBS vehicle werealso assessed. ADCs were dosed at 1 and 5 mg/kg three times over oneweek and thereafter monitored until an average tumor size ofapproximately 2000 mm³ was attained in the group administered withvehicle alone. At this point the Tumor Growth Inhibition was calculatedas described below.

Average tumor size relative to the vehicle treated group was calculatedas follows: tumors were measured with calipers twice a week until theaverage size of the vehicle group reached 1000 mm³; tumor size wascalculated using the formula (length×width²)/2. Tumor growth inhibitionwas calculated according to the following formula:(1−((T_(final)−T_(initial))/(C_(final)−C_(initial))))*100, where T(treated group) and C (control group) represent the mean tumor mass onthe day the vehicle group reached 1000 mm³. Results are summarized inTable 15.

TABLE 15 Final Tumor Average Tumor size at Day 8 Growth InhibitionTreatment Group mm³ (mean ± SD) (%) PBS Vehicle 2253 ± 217 0Control-MCC-DM1 1 mg/kg 2827 ± 278 −27 Control-MCC-DM1 5 mg/kg 2402 ±256 −7 Control-M0026 1 mg/kg 2729 ± 470 −22 Control-M0026 5 mg/kg 2787 ±503 −25 H1H1863N2-MCC-DM1 1 mg/kg  931 ± 292 62 H1H1863N2-MCC-DM1 5mg/kg  471 ± 227 84 H1H1863N2-M0026 1 mg/kg  679 ± 265 74H1H1863N2-M0026 5 mg/kg  96 ± 34 102

As summarized in Table 15, the greatest tumor inhibition was observed inmice dosed with 5 mg/kg H1H1863N2-M0026, where regression of the initialtumor was observed. The tumor growth inhibition of 102% resulting fromtreatment with 5 mg/kg H1H1863N2-M0026 was significantly greaterrelative to that observed following treatment of tumor with 5 mg/kgH1H1862N2-MCC-DM1 (83%). The superiority of the tumor growth inhibitioninduced by H1H1863N2-M0026 compared to H1H1863N2-mcc-DM1 was maintainedat the 1 mg/kg dose as well. No anti-tumor effect was observed in groupstreated with Control ADC using MCC-DM1 or M0026.

This Example therefore shows that anti-EGFRvIII antibodies of thepresent invention, when administered in the form of antibody-drugconjugates, are highly potent at inhibiting tumor growth. The presentExample additionally supports a role for the ADCs of the invention toactually promote tumor regression, especially in the context ofanti-EGFRvIII antibodies of the invention (e.g., H1H1863N2) conjugatedto the novel linker/drug molecule M0026.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

Example 13. Anti-EGFRvIII-DM1 Antibodies Show Specificity forEGFRvIII-Expressing Cells and Demonstrate Potent Cell Killing Activity

In this Example, the ability of anti-human EGFRvIII antibodiesconjugated to maytansine toxin DM1 to reduce cell viability wasdetermined using in vitro cell based assays.

Full length human EGFRvIII (SEQ ID NO:147) or wild-type human EGFR (SEQID NO:146) was stably introduced into HEK293 (293/hEGFRvIII,293/hEGFRwt), U251 (U251/hEGFRvIII) and MMT 060562 (MMT/hEGFRvIII) celllines. All cells were generated via lipofectamine 2000 basedmethodologies and were cultured in complete growth media in the presenceof G418.

Cell surface expression of EGFR wt or EGFRvIII was measured via FACSanalysis. Briefly, 1×10⁶ cells were incubated with 10 μg/ml ofanti-EGFRvIII antibody H1H1863N2, an anti-EGFRwt control mAb (ControlIV) or isotype control for 30 min. on ice in antibody dilution buffer.Following two washes with antibody dilution buffer, cells were incubatedwith 10 μg/ml of PE conjugated anti-human secondary antibody for 30 minon ice. After two additional washes, samples were run on an Accuri C6(BD) or Hypercyt (Intellicyt) cytometer and analyzed data analyzed usingFlowJo software. Results are summarized in Table 16. n.d.=notdetermined.

TABLE 16 Cell Surface Expression in EGFRwt and EGFRvIII Engineered CellLines FACS Binding (MFI Fold Above Isotype Control) H1H1863N2 Control IV(anti- (Anti- Secondary Isotype Cell Line Unstained EGFRvIII) EGFRwt)Alone Control HEK293 1X 1X  49x 1X 1X HEK293/ 1X n.d. 332x 1X 1X hEGFRwtHEK293/ 1X 264X n.d. 1X 1X hEGFRvIII U251 1X  1X n.d. 1X 1X U251/ 1X 13X n.d. 1X 1X hEGFRvIII MMT/ 1X  1X n.d. 1X 1X MMT/ 1X 280X n.d. 1X 1XhEGFRvIII

These results show that EGFRvIII surface expression was comparable inthe HEK293/hEGFRvIII and MMT/hEGFRvIII cells lines, whereasU251/EGFRvIII expression levels were approximately 20-fold lower than inthe HEK293/hEGFRvIII and MMT/hEGFRvIII cell systems. EGFRvIII bindingvia H1H1863N2 was not detectable in the parental cell lines. Incontrast, the anti-EGFRwt control antibody (Control IV) bound to HEK293parental cells at 49-fold above the isotype control. Stableincorporation of an EGFRwt expression vector into HEK293 cells increasedexpression to 332 fold above background and was comparable to EGFRvIIIexpression in HEK293/hEGFRvIII and MMT/hEGFRvIII cells.

The selective binding of anti-EGFRvIII antibody H1H1863N2 to EGFRvIIIwas assessed via FACS using HEK293 parental, HEK293/hEGFRwt,HEK293/hEGFRvIII, and A431 cell lines. Results are shown in Table 17.

TABLE 17 Binding Specificity of anti-EGFRvIII Antibody toEGFRvIII-Expressing Cell Lines FACS Binding (MFI Fold Above IsotypeControl) HEK293/ HEK293/ mAb HEK293 EGFRwt EGFRvIII A431 Control IV(Anti-EGFRwt) 83 251 855 621 H1H1863N2 (anti-EGFRvIII) 1 3 662 13Isotype Control 1 1 1 1 Secondary Ab Alone 1 1 1 1 Unstained Cells 1 1 11

As shown in Table 17, both H1H1863N2 and anti-EGFRwt control antibody(Control IV) exhibited strong binding (>650 fold above background) toHEK293/EGFRvIII cells relative to an isotype control. In contrast,H1H1863N2 bound weakly to the wt-EGFR HEK293 cell line (3-fold abovebackground) and endogenously expressing EGFR cell line A431 (13-foldabove control). Anti-EGFR-wt Control Antibody bound strongly to the wtEGFR-expressing cells, confirming the selectivity of H1H1863N2 forEGFRvIII over wild-type EGFR.

Next, the ability of anti-human EGFRvIII antibodies conjugated to themaytansine toxin DM1 to reduce cell viability was determined using invitro cell based assays. Cells were seeded in PDL-coated 96 well platesat 250-2000 cells per well in complete growth media and allowed to growovernight. For cell viability curves, ADCs or free drug (DM1-SMe) wasadded to cells at final concentrations ranging from 500 nM to 5 pM andincubated for 3 days. Cells were incubated with CCK8 (Dojindo) for thefinal 1-3 h and the absorbance at 450 nm (OD₄₅₀) was determined on theFlexstation3 (Molecular Devices). Background OD450 levels from digitonin(40 nM) treated cells was subtracted from all wells and viability isexpressed as a percentage of the untreated controls. IC50 values weredetermined from a four-parameter logistic equation over a 10-pointresponse curve (Graph Pad Prism). Results are shown in Tables 18A and18B. IC₅₀ values are in nM and are normalized for the particulardrug/antibody ratio (DAR).

TABLE 18A Cell Kill Potency of Anti-EGFRvIII-DM1 Antibody-DrugConjugates HEK293/ HEK293/ Cell Line HEK293 hEGFRvIII hEGFRwt U251 IC₅₀% IC₅₀ % IC₅₀ % IC₅₀ % ADC (nM) Kill (nM) Kill (nM) Kill (nM) KillH1H1863N2- >100 90 1 97 >100 91 48 77 MCC-DM1 Anti- 76 94 0.2 97 ~1.0 94ND ND EGFRwt- MCC-DM1 DM1-SMe 0.31 97 0.6 99 0.57 95 1.8 81 Isotype >10092 >100 96 >100 91 40 77 Control- MCC-DM1

TABLE 18B Cell Kill Potency of Anti-EGFRvIII-DM1 Antibody-DrugConjugates Cell Line U251/hEGFRvIII MMT MMT/hEGFRvIII IC₅₀ IC₅₀ IC₅₀ ADC(nM) % Kill (nM) % Kill (nM) % Kill H1H1863N2- 4 78 >150 40 3 100MCC-DM1 Anti- ND ND ND ND ND ND EGFRwt- MCC-DM1 DM1-SMe 1.2 83 0.6 960.7 100 Isotype 35 76 >150 66 NK 72 Control- MCC-DM1

As shown in Tables 18A and 18B, H1H1863N2-MCC-DM1 reduced the viabilityof HEK293/hEGFRvIII, U251/hEGFRvIII, and MMT/hEGFRvIII cell lines withIC50s ranging from 1.0 to 4.0 nM. In contrast, an isotype controlconjugated to DM1 reduced the viability of 293/EGFRvIII andMMT/hEGFRvIII cells with 1050s greater than 100 nM and U251/hEGFRvIIIcells with an IC50 of 35 nM. H1H1863N2-MCC-DM1 had no impact on HEK293cells expressing wild-type EGFR (293/hEGFRwt) or on the control parentalcell lines suggesting specificity for EGFRvIII expressing cells.

Thus, this Example demonstrates that the EGFRvIII antibody H1H1863N2 hasspecificity for EGFRvIII-expressing cell lines and demonstrates specificcell killing ability when conjugated to the DM1 toxin.

Example 14. Improved Cell Killing Potency is Achieved when an EGFRvIIIConformational-Binding Antibody-Drug Conjugate is Dosed in Combinationwith an EGFRvIII Junctional Peptide-Binding Antibody-Drug Conjugate

In this example, the ability to enhance cell killing by co-administeringtwo different types of anti-EGFRvIII antibody-drug conjugates wasdetermined. For this Example the combinations tested consisted of twodifferent anti-EGFRvIII antibodies: (1) an anti-EGFRvIII specificantibody that does not recognize the EGFRvIII junctional peptide ADC(referred to herein as a “conformational binder”); and (2) ananti-EGFRvIII specific antibody that does recognize the EGFRvIIIjunctional peptide (referred to herein as a “peptide binder”). Asdemonstrated in Example 6, the anti-EGFRvIII antibody H1H1863N2 does notbind to the EGFRvIII junctional peptide or residues 311-326 of humanEGFR and is therefore regarded as a “conformational binder”.

Cross Competition In Vitro

First, the ability of H1H1863N2 to cross compete with an antibody thatbinds the EGFRvIII junctional peptide was determined via a bindingcompetition assay. The junctional peptide binding anti-EGFRvIII antibodyused in this example was Control V.

Cross competition was determined using a real time, label-free bio-layerinterferometry (BLI) assay on an Octet HTX biosensor (ForteBio Corp., ADivision of Pall Life Sciences). The entire experiment was performed at25° C. in buffer comprised of 0.01M HEPES pH7.4, 0.15M NaCl, 3 mM EDTA,0.05% v/v Surfactant P20, 1.0 mg/mL BSA (Octet HBST buffer) with theplate shaking at a speed of 1000 rpm. To assess whether two antibodiescross-competed for binding on recombinant human EGFRvIII (hEGFRvIII.mmh;SEQ ID:152), approximately ˜0.35 nm of hEGFRvIII.mmh was captured ontoanti-penta-His coated Octet biosensors. The antigen-captured biosensorswere then saturated with the first anti-EGFRvIII monoclonal antibody(subsequently referred to as mAb-1) by immersion into wells containing a50 μg/mL solution of mAb-1 for 5 minutes. The biosensors were thensubsequently submerged into wells containing a 50 μg/mL solution of asecond anti-EGFRvIII monoclonal antibody (subsequently referred to asmAb-2) for 3 minutes. All the biosensors were washed in Octet HBSTbuffer in between each step of the experiment. The real-time bindingresponse was monitored during the course of the experiment and thebinding response at the end of every step was recorded. The response ofmAb-2 binding to hEGFRvIII pre-complexed with mAb-1 was compared andcompetitive/non-competitive behavior of different anti-EGFRvIIImonoclonal antibodies was determined.

Using this experimental cross-competition format, H1H1863N2 did notexhibit cross competition with the EGFRvIII junctional peptide bindertested, nor did it cross compete for binding to EGFRvIII with Control IIor Control IV. The results of this cross competition assay thereforeindicate that H1H1863N2 has a distinct binding epitope to that of theEGFRvIII junctional peptide binder, as well as Controls II and IV.

Cell Killing Activity of Individual Anti-EGFRvIII Antibody-DrugConjugates

Next, the ability of H1H1863N2-MCC-DM1 and an anti-EGFRvIIIpeptide-binding ADC to reduce cell viability when administered incombination was assessed. The ability of Control V to induce cell killwhen conjugated to SMCC-DM1 (i.e., Control V-MCC-DM1) was determinedusing an in vitro cell based assay as described in Example 13. Resultsare summarized in Table 19.

TABLE 19 Cell Kill Potency of Anti-EGFRvIII-DM1 Antibody-Drug ConjugatesHEK293/ MMT/ hEGFRvIII hEGFRvIII Cell Line HEK293 (high) MMT (high) % %% % ADC IC50 Kill IC50 Kill IC50 Kill IC50 Kill DM1-SMe 0.19 98 0.25 990.15 100 0.18 99 (free DM1) Isotype 200 91 150 92 110 68 250 72 Ctrl-MCC-DM1 H1H1863N2- 80 97 0.37 99 200 95 3.25 97 MCC-DM1 Control V- 90 950.25 100 200 89 0.35 97 MCC-DM1

As summarized in Table 19, anti-EGFRvIII ADCs reduced cell viability ofvarious EGFRvIII overexpressing cell lines with IC₅₀ values ranging from0.25 nM to 3.25 nM.

Cell Killing Activity of Pairwise Combinations of Anti-EGFRvIIIAntibody-Drug Conjugates

Next, the cell killing potency of H1H1863N2-MCC-DM1 paired with theanti-EGFRvIII peptide-binding ADC was tested on EGFRvIII over-expressingcell lines in a 1:1 ratio. Results are shown in Table 20.

TABLE 20 Cell Kill Potency of Pairwise Combinations of Anti-EGFRvIII-DM1ADCs HEK293/ MMT/ Cell Line: HEK293 hEGFRvIII MMT hEGFRvIII IC₅₀ IC₅₀ %IC₅₀ IC₅₀ ADC 1 ADC 2 (nM) % Kill (nM) Kill (nM) % Kill (nM) % KillH1H1863N2- None 250 87 1.52 95 250 59 11.1 98 MCC-DM1 Control V- None100 85 0.14 98 100 67 0.7 95 MCC-DM1 H1H1863N2- Control 100 91 0.19 99200 98 0.58 100 MCC-DM1 V- MCC- DM1 DM1-SMe None 0.21 96 0.28 97 0.19100 0.19 100 (Free DM1) Isotype Ctrl- None 200 93 95 93 150 32 100 36MCC-DM1

As summarized in Table 20, the combination of H1H1863N2-MCC-DM1 (aconformational epitope binder) and the Control V-MCC-DM1 (a junctionalpeptide binder) resulted in cell killing potency that was at leastequivalent to, or in certain instances, enhanced as compared with thesingle-ADC treatments. The lack of interference between the two types ofantibodies suggests the effective use of two non-competing antibodieswith different cytotoxins, or different classes of cytotoxins havingdistinct mechanisms of action.

In summary, this example demonstrates that H1H1863N2 does notcross-compete with the control EGFRvIII peptide binding antibody. Thisunique epitope allows for its combination with EGFRvIII peptide-bindingADCs to improve cell killing potency. This novel combination of EGFRvIIIADCs may allow for better therapeutic efficacy.

What is claimed is:
 1. A method for treating a cancer or tumorexpressing EGFRvIII, comprising administering to a subject in needthereof a therapeutically effective amount of a pharmaceuticalcomposition comprising an isolated antibody or antigen-binding fragmentthereof that specifically binds human EGFRvIII, but binds neither thejunctional peptide of SEQ ID NO:148 nor the peptide of SEQ ID NO:165,wherein the antibody comprises the complementarity determining regions(CDRs) within the heavy chain variable region/light chain variableregion (HCVR/LCVR) amino acid sequence pair of SEQ ID NOs: 34/42, and apharmaceutically acceptable carrier, wherein the cancer or tumor growthis inhibited.
 2. The method of claim 1, wherein the cancer or tumor isselected from the group consisting of glioblastoma, ductal orintraductal breast carcinoma, non-small cell lung carcinomas, ovariancarcinomas, prostate cancer, and squamous cell carcinoma of the head andneck.
 3. The method of claim 1, further comprising treating the subjectwith one or more additional therapeutic agents selected from the groupconsisting of a chemotherapeutic agent, anti-inflammatory agents, andanalgesics.
 4. The method of claim 1, wherein the HCVR comprises anamino acid sequence of SEQ ID NO:
 34. 5. The method of claim 1, whereinthe LCVR comprises an amino acid sequence of SEQ ID NO:
 42. 6. Themethod of claim 1, wherein the antibody or antigen-binding fragmentcomprises a HCVR/LCVR sequence pair of SEQ ID NO: 34/42.
 7. The methodof claim 1, wherein the antibody or antigen-binding fragment isconjugated to a cytotoxin.
 8. The method of claim 7, wherein thecytotoxin is selected from the group consisting of biotoxins,chemotherapeutic agents and radioisotopes.
 9. The method of claim 7,wherein the cytotoxin is selected from the group consisting ofmaytansinoids, auristatins, tomaymycins, duocarmycins, 225Ac, 227Th, andany derivatives thereof.
 10. The method of claim 9, wherein the antibodyor antigen-binding fragment is conjugated to a maytansinoid.
 11. Amethod for treating a cancer, reducing tumor growth and/or causing tumorregression in a patient, the method comprising administering to apatient in need thereof a first antibody-drug conjugate (ADC) comprisingan antibody or antigen-binding fragment thereof wherein the antibodycomprises the CDRs within the HCVR/LCVR amino acid sequence pair of SEQID NOs: 34/42, and a cytotoxin, wherein the antibody or antigen-bindingfragment of the first ADC specifically binds EGFRvIII but binds neitherthe junctional peptide of SEQ ID NO:148 nor the peptide of SEQ IDNO:165, wherein the cancer or tumor growth is inhibited or the tumorregresses.
 12. The method of claim 11, wherein the method furthercomprises administering to the patient a second ADC comprising anantibody or antigen-binding fragment thereof and a cytotoxin, whereinthe antibody or antigen-binding fragment of the second ADC specificallybinds EGFRvIII and also binds the junctional peptide of SEQ ID NO:148and/or the peptide of SEQ ID NO:165.
 13. The method of claim 11, whereinthe antibody or antigen-binding fragment of the first ADC comprises aheavy chain variable region comprising SEQ ID NO: 34 and a light chainvariable region comprising SEQ ID NO: 42.